Antisense modulation of PPAR-delta expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of PPAR-delta. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding PPAR-delta. Methods of using these compounds for modulation of PPAR-delta expression and for treatment of diseases associated with expression of PPAR-delta are provided.

INTRODUCTION

[0001] This application is a continuation of U.S. Ser. No. 10/160,807 filed May 31, 2002, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention provides compositions and methods for modulating the expression of PPAR-delta. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding PPAR-delta. Such compounds have been shown to modulate the expression of PPAR-delta.

BACKGROUND OF THE INVENTION

[0003] Steroid, thyroid and retinoid hormones produce a diverse array of physiologic effects through the regulation of gene expression. Upon entering the cell, these hormones bind to a unique group of intracellular nuclear receptors which have been characterized as ligand-dependent transcription factors. This complex then moves into the nucleus where the receptor and its cognate ligand interact with the transcription preinitiation complex affecting its stability and ultimately the rate of transcription of the target genes.

[0004] The Peroxisome Proliferator-Activated Receptors (PPARs) are members of the nuclear hormone receptor subfamily of transcription factors. PPARs form heterodimers with other members of the nuclear hormone receptor superfamily and these heterodimers regulate the transcription of various genes. There are 3 known subtypes of PPARs, PPAR-alpha, PPAR-delta (also known as NUC1, PPAR-beta and FAAR) and two isoforms of PPAR-gamma.

[0005] PPAR-alpha is expressed mostly in brown adipose tissue and liver while PPAR-gamma is mainly expressed in adipose and to a lesser extent in the colon. PPAR-delta is found in many tissue with the highest expression in the gut, kidney and heart. Because of their localization to adipose, PPAR-alpha and PPAR-gamma have received the most attention. However, recently it has been shown that PPAR-delta is localized to both skeletal muscle and fat but as ligands that activate PPAR-delta do not affect glucose or lipid concentrations, the role of PPAR-delta in skeletal muscle is unclear (Berger et al., The Journal of Blological Chemistry, 1999, 274, 6718-6725; Loviscach et al., Diabetologia, 2000, 43, 304-311). PPAR-delta has recently been connected with the clinical manifestations of colon cancer (Gupta et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 13275-13280; He et al., Cell, 1999, 99, 335-345). It has also been shown to play a role in the regulation of the expression of acyl-CoA synthetase 2 in the brain and lipid metabolism (Basu-Modak et al., J. Biol. Chem., 1999, 274, 35881-35888), repression of other PPAR and thyroid receptors (Jow and Mukherjee, J. Biol. Chem., 1995, 270, 3836-3840) as well as embryo implantation and decidualization (Lim and Dey, Trends Endocrinol. Metab., 2000, 11, 137-142; Lim et al., Genes Dev., 1999, 13, 1561-1574).

[0006] PPAR-delta was first isolated from a human osteosarcoma cell library (SAOS-2/ B10) and shown to be activated by fatty acids (Schmidt et al., Mol. Endocrinol., 1992, 6, 1634-1641). It was subsequently cloned by Amri et al. from a preadipocyte library and implicated as a likely mediator of fatty acid transcriptional effects in preadipocytes (Amri et al., J. Biol. Chem., 1995, 270, 2367-2371). Disclosed in U.S. Pat. No. 5,861,274 and the corresponding PCT Publication WO 96/01317 are the nucleic acid and protein sequences of human PPAR-delta (Evans et al., 1996; Evans et al., 1999).

[0007] Mano et al. cloned the rabbit PPAR-delta gene from mature rabbit osteoclasts and demonstrated that carbaprostacyclin-induced bone resorption could be blocked by a phosphorothioate antisense oligonucleotide (21-mer spanning the start codon) targeting rabbit PPAR-delta (Mano et al., J. Biol. Chem., 2000, 275, 8126-8132) suggesting a role for PPAR-delta in bone metabolism.

[0008] Assignment of the PPAR-delta gene to human chromosome 6p21 places it in a region of disease genes previously mapped to chromosome 6 (Yoshikawa et al., Genomics, 1996, 35, 637-638).

[0009] The pharmacological modulation of PPAR-delta activity and/or expression is therefore believed to be an appropriate point of therapeutic intervention in pathological conditions such as cancer, osteoporosis, diabetes and various endocrine disorders.

[0010] Currently, there are no known therapeutic agents which effectively inhibit the synthesis of PPAR-delta. Consequently, there remains a long felt need for agents capable of effectively inhibiting PPAR-delta function.

[0011] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of PPAR-delta expression.

[0012] The present invention provides compositions and methods for modulating PPAR-delta expression.

SUMMARY OF THE INVENTION

[0013] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding PPAR-delta, and which modulate the expression of PPAR-delta. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of PPAR-delta in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of PPAR-delta by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding PPAR-delta, ultimately modulating the amount of PPAR-delta produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding PPAR-delta. As used herein, the terms “target nucleic acid” and “nucleic acid encoding PPAR-delta” encompass DNA encoding PPAR-delta, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of PPAR-delta. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.

[0015] It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding PPAR-delta. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding PPAR-delta, regardless of the sequence(s) of such codons.

[0016] It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon.

[0017] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an MRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.

[0018] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It has also been found that introns can be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

[0019] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and extronic regions.

[0020] Upon excision of one or more exon or intron regions or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0021] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites.

[0022] Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0023] In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.

[0024] An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

[0025] Antisense and other compounds of the invention, which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The sites to which these preferred antisense compounds are complementary are hereinbelow referred to as “preferred target regions” and are therefore preferred sites for targeting. As used herein the term “preferred target region” is defined as at least an 8-nucleobase portion of a region of a gene that is accessible for hybridization with a complementary sequence of nucleic acid.

[0026] While the specific sequences of particular preferred target regions can be represented by the reverse complement of the antisense oligonucleotides set forth in Table 1, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target regions may be identified by one having ordinary skill.

[0027] Stretches of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target regions are considered to be suitable preferred target regions as well. Also, stretches of DNA or RNA that are about 8 to about 80 consecutive nucleobases and that comprise some portion of the 5′- or 3′-terminal sequence of a preferred target region will also be considered preferred target region for purposes of this invention. Exemplary good preferred target regions include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one a preferred target region (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the preferred target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly good preferred target regions are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of a preferred target region (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 3′-terminus of the preferred target region and continuing until the target site contains about 8 to about 80 nucleobases). One having skill in the art, once armed with the empirically-derived preferred target regions herein will be able, without undue experimentation, to identify further preferred target regions. In addition, one having ordinary skill in the art will also be able to identify additional compounds, including oligonucleotide probes and primers, that hybridize to these preferred target regions using techniques available to the ordinary practitioner in the art.

[0028] Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.

[0029] For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0030] Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0031] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0032] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.

[0033] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[0034] While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides from about 8 to about 50 nucleobases, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.

[0035] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0036] Exemplary preferred antisense compounds include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art, once armed with the empirically-derived preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.

[0037] Antisense and other compounds of the invention, which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are herein identified as preferred embodiments of the invention. While specific sequences of the antisense compounds are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred antisense compounds may be identified by one having ordinary skill.

[0038] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. In addition, linear structures may also have internal nucleobase complementarity and may therefore fold in a manner as to produce a double stranded structure. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0039] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0040] Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0041] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0042] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0043] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0044] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0045] Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃) —CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O —P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0046] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylamino-ethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0047] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′—OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂-CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0048] A further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH₂—)n group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0049] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[31,21:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyl-adenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0050] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0051] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, .660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Phariacol. Exp. Ther., 1996, 277, 923-937). Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. Patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0052] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0053] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as interferon-induced RNAseL which cleaves both cellular and viral RNA. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0054] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0055] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0056] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0057] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0058] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0059] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

[0060] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.

[0061] For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

[0062] The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of PPAR-delta is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.

[0063] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding PPAR-delta, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding PPAR-delta can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of PPAR-delta in a sample may also be prepared.

[0064] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

[0065] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.

[0066] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. applications Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.

[0067] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0068] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0069] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0070] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0071] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

[0072] Emulsions

[0073] The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

[0074] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories:

[0075] synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0076] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

[0077] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[0078] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0079] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[0080] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0081] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

[0082] In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0083] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[0084] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

[0085] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

[0086] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0087] Liposomes

[0088] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

[0089] Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

[0090] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

[0091] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0092] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

[0093] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

[0094] Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.

[0095] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0096] Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0097] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0098] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0099] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/ cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0100] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765). Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1)or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

[0101] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C₁₂15G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[0102] A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.

[0103] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0104] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0105] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0106] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0107] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0108] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0109] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0110] Penetration Enhancers

[0111] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[0112] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

[0113] Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0114] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0115] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydrofusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0116] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0117] Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacycloalkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0118] Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.

[0119] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

[0120] Carriers

[0121] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0122] Excipients

[0123] In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0124] Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0125] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

[0126] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0127] Other Components

[0128] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0129] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0130] Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0131] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0132] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0133] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-Alkoxy Amidites

[0134] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2′-alkoxy amidites, optimized synthesis cycles were developed that incorporate multiple steps coupling longer wait times relative to standard synthesis cycles.

[0135] The following abbreviations are used in the text: thin layer chromatography (TLC), melting point (MP), high pressure liquid chromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar), methanol (MeOH), dichloromethane (CH₂Cl₂), triethylamine (TEA), dimethyl formamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF).

[0136] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC) nucleotides were synthesized according to published methods (Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.) or prepared as follows:

[0137] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for 5-Methyl dC Amidite

[0138] To a 50 L glass reactor equipped with air stirrer and Ar gas line was added thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) at ambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol, 1.05 eq) was added as a solid in four portions over 1 h. After 30 min, TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent and by-products and 2% 3′,5′-bis DMT product (R_(f) in EtOAc 0.45, 0.05, 0.98, 0.95 respectively). Saturated sodium bicarbonate (4 L) and CH₂Cl₂ were added with stirring (pH of the aqueous layer 7.5). An additional 18 L of water was added, the mixture was stirred, the phases were separated, and the organic layer was transferred to a second 50 L vessel. The aqueous layer was extracted with additional CH₂Cl₂ (2×2 L). The combined organic layer was washed with water (10 L) and then concentrated in a rotary evaporator to approx. 3.6 kg total weight. This was redissolved in CH₂Cl₂ (3.5 L), added to the reactor followed by water (6 L) and hexanes (13 L). The mixture was vigorously stirred and seeded to give a fine white suspended solid starting at the interface. After stirring for 1 h, the suspension was removed by suction through a ½″ diameter teflon tube into a 20 L suction flask, poured onto a 25 cm Coors Buchner funnel, washed with water (2×3 L) and a mixture of hexanes-CH₂Cl₂ (4:1, 2×3 L) and allowed to air dry overnight in pans (1″ deep). This was further dried in a vacuum oven (75° C., 0.1 mm Hg, 48 h) to a constant weight of 2072 g (93%) of a white solid, (mp 122-124° C.). TLC indicated a trace contamination of the bis DMT product. NMR spectroscopy also indicated that 1-2 mole percent pyridine and about 5 mole percent of hexanes was still present.

[0139] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine Intermediate for 5-Methyl-dC Amidite

[0140] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and an Ar gas line was added 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrous acetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C.). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30 minutes while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition. The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R_(f) 0.43 to 0.84 of starting material and silyl product, respectively). Upon completion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60 min so as to maintain the temperature between −20° C. and −10° C. during the strongly exothermic process, followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1 h. TLC indicated a complete conversion to the triazole product (R_(f) 0.83 to 0.34 with the product spot glowing in long wavelength UV light). The reaction mixture was a peach-colored thick suspension, which turned darker red upon warming without apparent decomposition. The reaction was cooled to −15° C. internal temperature and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The combined water layers were back-extracted with EtOAc (6 L). The water layer was discarded and the organic layers were concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The second half of the reaction was treated in the same way. Each residue was dissolved in dioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight (although the reaction is complete within 1 h).

[0141] TLC indicated a complete reaction (product R_(f) 0.35 in EtOAc-MeOH 4:1). The reaction solution was concentrated on a rotary evaporator to a dense foam. Each foam was slowly redissolved in warm EtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, and extracted with water (2×4L) to remove the triazole by-product. The water was back-extracted with EtOAc (2 L). The organic layers were combined and concentrated to about 8 kg total weight, cooled to 0° C. and seeded with crystalline product. After 24 hours, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L) until a white powder was left and then washed with ethyl ether (2×3L). The solid was put in pans (1″ deep) and allowed to air dry overnight. The filtrate was concentrated to an oil, then redissolved in EtOAc (2 L), cooled and seeded as before. The second crop was collected and washed as before (with proportional solvents) and the filtrate was first extracted with water (2×1L) and then concentrated to an oil. The residue was dissolved in EtOAc (1 L) and yielded a third crop which was treated as above except that more washing was required to remove a yellow oily layer.

[0142] After air-drying, the three crops were dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g, respectively) and combined to afford 2550 g (85%) of a white crystalline product (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity. The mother liquor still contained mostly product (as determined by TLC) and a small amount of triazole (as determined by NMR spectroscopy), bis DMT product and unidentified minor impurities. If desired, the mother liquor can be purified by silica gel chromatography using a gradient of MeOH (0-25%) in EtOAc to further increase the yield.

[0143] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine Penultimate Intermediate for 5-Methyl dC Amidite

[0144] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000 g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambient temperature in a 50 L glass reactor vessel equipped with an air stirrer and argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86 mol, 1.05 eq) was added and the reaction was stirred at ambient temperature for 8 h. TLC (CH₂Cl₂-EtOAc; CH₂Cl₂-EtOAc 4:1; R_(f) 0.25) indicated approx. 92% complete reaction. An additional amount of benzoic anhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLC indicated approx. 96% reaction completion. The solution was diluted with EtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added with stirring, and the mixture was extracted with water (15 L, then 2×10 L). The aqueous layer was removed (no back-extraction was needed) and the organic layer was concentrated in 2×20 L rotary evaporator flasks until a foam began to form. The residues were coevaporated with acetonitrile (1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a dense foam. High pressure liquid chromatography (HPLC) revealed a contamination of 6.3% of N4, 3′-O-dibenzoyl product, but very little other impurities.

[0145] The product was purified by Biotage column chromatography (5 kg Biotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L). The crude product (800 g),dissolved in CH₂Cl₂ (2 L), was applied to the column. The column was washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1 solvent mixture (10 kg), then 99:1 EtOAc:TEA (17 kg). The fractions containing the product were collected, and any fractions containing the product and impurities were retained to be resubjected to column chromatography. The column was re-equilibrated with the original 65:35:1 solvent mixture (17 kg). A second batch of crude product (840 g) was applied to the column as before. The column was washed with the following solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1 (10 kg), and 99:1 EtOAc:TEA(15 kg). The column was reequilibrated as above, and a third batch of the crude product (850 g) plus impure fractions recycled from the two previous columns (28 g) was purified following the procedure for the second batch. The fractions containing pure product combined and concentrated on a 20L rotary evaporator, co-evaporated with acetontirile (3 L) and dried (0.1 mm Hg, 48 h, 25° C.) to a constant weight of 2023 g (85%) of white foam and 20 g of slightly contaminated product from the third run. HPLC indicated a purity of 99.8% with the balance as the diBenzoyl product.

[0146] [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-Methyl dC Amidite)

[0147] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidine (998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (300 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (15 ml) was added and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2.5 L) and water (600 ml), and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (7.5 L) and hexane (6 L). The two layers were separated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L) and water (3×2 L), and the phases were separated. The organic layer was dried (Na₂SO₄), filtered and rotary evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried to a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g an off-white foam solid (96%).

[0148] 2′-Fluoro Amidites

[0149] 2′-Fluorodeoxyadenosine Amidites

[0150] 2′-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. The preparation of 2′- fluoropyrimidines containing a 5-methyl substitution are described in U.S. Pat. No. 5,861,493. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and whereby the 2′-alpha-fluoro atom is introduced by a SN2-displacement of a 2′-beta-triflate group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0151] 2′-Fluorodeoxyguanosine

[0152] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate isobutyryl-arabinofuranosylguanosine. Alternatively, isobutyryl-arabinofuranosylguanosine was prepared as described by Ross et al., (Nucleosides & Nucleosides, 16, 1645, 1997). Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give isobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.

[0153] 2′-Fluorouridine

[0154] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a literature procedure in which 2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′ phosphoramidites.

[0155] 2′-Fluorodeoxycytidine

[0156] 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′ phosphoramidites.

[0157] 2′-O-(2-Methoxyethyl) Modified Amidites

[0158] 2′-O-Methoxyethyl-substituted nucleoside amidites (otherwise known as MOE amidites) are prepared as follows, or alternatively, as per the methods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).

[0159] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate

[0160] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol), tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60 g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12 L three necked flask and heated to 130° C. (internal temp) at atmospheric pressure, under an argon atmosphere with stirring for 21 h. TLC indicated a complete reaction. The solvent was removed under reduced pressure until a sticky gum formed (50-85° C. bath temp and 100-11 mm Hg) and the residue was redissolved in water (3 L) and heated to boiling for 30 min in order the hydrolyze the borate esters. The water was removed under reduced pressure until a foam began to form and then the process was repeated. HPLC indicated about 77% product, 15% dimer (5′ of product attached to 2′ of starting material) and unknown derivatives, and the balance was a single unresolved early eluting peak.

[0161] The gum was redissolved in brine (3 L), and the flask was rinsed with additional brine (3 L). The combined aqueous solutions were extracted with chloroform (20 L) in a heavier-than continuous extractor for 70 h. The chloroform layer was concentrated by rotary evaporation in a 20 L flask to a sticky foam (2400 g). This was coevaporated with MeOH (400 mL) and EtOAc (8 L) at 75° C. and 0.65 atm until the foam dissolved at which point the vacuum was lowered to about 0.5 atm. After 2.5 L of distillate was collected a precipitate began to form and the flask was removed from the rotary evaporator and stirred until the suspension reached ambient temperature. EtOAc (2 L) was added and the slurry was filtered on a 25 cm table top Buchner funnel and the product was washed with EtOAc (3×2 L). The bright white solid was air dried in pans for 24 h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to afford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).

[0162] The brine layer in the 20 L continuous extractor was further extracted for 72 h with recycled chloroform. The chloroform was concentrated to 120 g of oil and this was combined with the mother liquor from the above filtration (225 g), dissolved in brine (250 mL) and extracted once with chloroform (250 mL). The brine solution was continuously extracted and the product was crystallized as described above to afford an additional 178 g of crystalline product containing about 2% of thymine. The combined yield was 1827 g (69.4%). HPLC indicated about 99.5% purity with the balance being the dimer.

[0163] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine Penultimate Intermediate

[0164] In a 50 L glass-lined steel reactor, 2′-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol), lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile (15 L). The solution was stirred rapidly and chilled to −10° C. (internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g, 5.21 mol) was added as a solid in one portion. The reaction was allowed to warm to −2° C. over 1 h. (Note: The reaction was monitored closely by TLC (EtOAc) to determine when to stop the reaction so as to not generate the undesired bis-DMT substituted side product). The reaction was allowed to warm from −2 to 3° C. over 25 min. then quenched by adding MeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L). The solution was transferred to a clear 50 L vessel with a bottom outlet, vigorously stirred for 1 minute, and the layers separated. The aqueous layer was removed and the organic layer was washed successively with 10% aqueous citric acid (8 L) and water (12 L). The product was then extracted into the aqueous phase by washing the toluene solution with aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueous layer was overlayed with toluene (12 L) and solid citric acid (8 moles, 1270 g) was added with vigorous stirring to lower the pH of the aqueous layer to 5.5 and extract the product into the toluene. The organic layer was washed with water (10 L) and TLC of the organic layer indicated a trace of DMT-O-Me, bis DMT and dimer DMT.

[0165] The toluene solution was applied to a silica gel column (6 L sintered glass funnel containing approx. 2 kg of silica gel slurried with toluene (2 L) and TEA(25 mL)) and the fractions were eluted with toluene (12 L) and EtOAc (3×4 L) using vacuum applied to a filter flask placed below the column. The first EtOAc fraction containing both the desired product and impurities were resubjected to column chromatography as above. The clean fractions were combined, rotary evaporated to a foam, coevaporated with acetonitrile (6 L) and dried in a vacuum oven (0.1 mm Hg, 40 h, 40° C.) to afford 2850 g of a white crisp foam. NMR spectroscopy indicated a 0.25 mole % remainder of acetonitrile (calculates to be approx. 47 g) to give a true dry weight of 2803 g (96%). HPLC indicated that the product was 99.41% pure, with the remainder being 0.06 DMT-O-Me, 0.10 unknown, 0.44 bis DMT, and no detectable dimer DMT or 3′-O-DMT.

[0166] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T Amidite)

[0167] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine (1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solution was co-evaporated with toluene (200 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g, 1.0 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (20 ml) was added and the solution was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (3.5 L) and water (600 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.6 L) and extracted with the mixture of toluene (12 L) and hexanes (9 L). The upper layer was washed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organic layer was dried (Na₂SO₄), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of an off-white foamy solid (95%).

[0168] Preparation of 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate

[0169] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and argon gas line was added 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine (2.616 kg, 4.23 mol, purified by base extraction only and no scrub column), anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C.). Trimethylsilylchloride (1.60 L, 12.7 mol, 3.0 eq) was added over 30 min. while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). (Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition). The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc, R_(f) 0.68 and 0.87 for starting material and silyl product, respectively). Upon completion, triazole (2.34 kg, 33.8 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was added slowly over 60 min so as to maintain the temperature between −20° C. and −10° C. (note: strongly exothermic), followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1 h, at which point it was an off-white thick suspension. TLC indicated a complete conversion to the triazole product (EtOAc, R_(f) 0.87 to 0.75 with the product spot glowing in long wavelength UV light). The reaction was cooled to −15° C. and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The second half of the reaction was treated in the same way. The combined aqueous layers were back-extracted with EtOAc (8 L) The organic layers were combined and concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The residue was dissolved in dioxane (2 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight TLC indicated a complete reaction (CH₂Cl₂-acetone-MeOH, 20:5:3, R_(f) 0.51). The reaction solution was concentrated on a rotary evaporator to a dense foam and slowly redissolved in warm CH₂Cl₂ (4 L, 40° C.) and transferred to a 20 L glass extraction vessel equipped with a air-powered stirrer. The organic layer was extracted with water (2×6 L) to remove the triazole by-product. (Note: In the first extraction an emulsion formed which took about 2 h to resolve). The water layer was back-extracted with CH₂Cl₂ (2×2 L), which in turn was washed with water (3 L). The combined organic layer was concentrated in 2×20 L flasks to a gum and then recrystallized from EtOAc seeded with crystalline product. After sitting overnight, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc until a white free-flowing powder was left (about 3×3 L). The filtrate was concentrated to an oil recrystallized from EtOAc, and collected as above. The solid was air-dried in pans for 48 h, then further dried in a vacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a bright white, dense solid (86%). An HPLC analysis indicated both crops to be 99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAc remained.

[0170] Preparation of 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidine Penultimate Intermediate:

[0171] Crystalline 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine (1000 g, 1.62 mol) was suspended in anhydrous DMF (3 kg) at ambient temperature and stirred under an Ar atmosphere. Benzoic anhydride (439.3 g, 1.94 mol) was added in one portion. The solution clarified after 5 hours and was stirred for 16 h. HPLC indicated 0.45% starting material remained (as well as 0.32% N4, 3′-O-bis Benzoyl). An additional amount of benzoic anhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLC indicated no starting material was present. TEA (450 mL, 3.24 mol) and toluene (6 L) were added with stirring for 1 minute. The solution was washed with water (4×4 L), and brine (2×4 L). The organic layer was partially evaporated on a 20 L rotary evaporator to remove 4 L of toluene and traces of water. HPLC indicated that the bis benzoyl side product was present as a 6% impurity. The residue was diluted with toluene (7 L) and anhydrous DMSO (200 mL, 2.82 mol) and sodium hydride (60% in oil, 70 g, 1.75 mol) was added in one portion with stirring at ambient temperature over 1 h. The reaction was quenched by slowly adding then washing with aqueous citric acid (10%, 100 mL over 10 min, then 2×4 L), followed by aqueous sodium bicarbonate (2%, 2 L), water (2×4 L) and brine (4 L). The organic layer was concentrated on a 20 L rotary evaporator to about 2 L total volume. The residue was purified by silica gel column chromatography (6 L Buchner funnel containing 1.5 kg of silica gel wetted with a solution of EtOAc-hexanes-TEA(70:29:1)). The product was eluted with the same solvent (30 L) followed by straight EtOAc (6 L). The fractions containing the product were combined, concentrated on a rotary evaporator to a foam and then dried in a vacuum oven (50° C., 0.2 mm Hg, 8 h) to afford 1155 g of a crisp, white foam (98%). HPLC indicated a purity of >99.7%.

[0172] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C Amidite)

[0173] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidine (1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporated with toluene (300 ml) at 50° C. under reduced pressure. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (9 L) and hexanes (6 L). The two layers were separated and the upper layer was washed with DMF-water (60:40 v/v, 3×3 L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1336 g of an off-white foam (97%).

[0174] Preparation of [51-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A Amdite)

[0175] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosine (purchased from Reliable Biopharmaceutical, St. Lois, Mo.), 1098 g, 1.5 mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene (300 ml) at 50 ° C. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (1.4 L) and extracted with the mixture of toluene (9 L) and hexanes (6 L). The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered and evaporated to a sticky foam. The residue was co-evaporated with acetonitrile (2.5 L) under reduced pressure and dried in a vacuum oven (25 ° C., 0.1 mm Hg, 40 h) to afford 1350 g of an off-white foam solid (96%).

[0176] Prepartion of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G Amidite)

[0177] 5′-O-(4,4′-dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrlguanosine (purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (200 ml) at 50° C., cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2 L) and water (600 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (2 L) and extracted with a mixture of toluene (10 L) and hexanes (5 L). The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and the solution was washed with water (3×4 L). The organic layer was dried (Na₂SO₄), filtered and evaporated to approx. 4 kg. Hexane (4 L) was added, the mixture was shaken for 10 min, and the supernatant liquid was decanted. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1660 g of an off-white foamy solid (91%).

[0178] 2′-O-(Aminooxyethyl) Nucleoside Amidites and 2′-O-(dimethylaminooxyethyl) Nucleoside Amidites

[0179] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

[0180] 2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known in the art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.

[0181] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0182] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22, EtOAc) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between CH₂Cl₂ (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether (600 mL) and cooling the solution to −10° C. afforded a white crystalline solid which was collected by filtration, washed with ethyl ether (3×2 00 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g of white solid (74.8%). TLC and NMR spectroscopy were consistent with pure product.

[0183] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0184] In the fume hood, ethylene glycol (350 mL, excess) was added cautiously with manual stirring to a 2 L stainless steel pressure reactor containing borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). (Caution : evolves hydrogen gas). 5′-O-tert-Butyldiphenylsilyl-O2-2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160 ° C. was reached and then maintained for 16 h (pressure<100 psig). The reaction vessel was cooled to ambient temperature and opened. TLC (EtOAc, R_(f) 0.67 for desired product and R_(f) 0.82 for ara-T side product) indicated about 70% conversion to the product. The solution was concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. (Alternatively, once the THF has evaporated the solution can be diluted with water and the product extracted into EtOAc). The residue was purified by column chromatography (2 kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, evaporated and dried to afford 84 g of a white crisp foam (50%), contaminated starting material (17.4 g, 12% recovery) and pure reusable starting material (20 g, 13% recovery). TLC and NMR spectroscopy were consistent with 99% pure product.

[0185] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0186] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5- methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol) and dried over P₂O₅ under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle). Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture with the rate of addition maintained such that the resulting deep red coloration is just discharged before adding the next drop. The reaction mixture was stirred for 4 hrs., after which time TLC (EtOAc:hexane, 60:40) indicated that the reaction was complete. The solvent was evaporated in vacuuo and the residue purified by flash column chromatography (eluted with 60:40 EtOAc:hexane), to yield 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%) upon rotary evaporation.

[0187] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0188] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate washed with ice cold CH₂Cl₂, and the combined organic phase was washed with water and brine and dried (anhydrous Na₂SO₄). The solution was filtered and evaporated to afford 2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was stirred for 1 h. The solvent was removed under vacuum and the residue was purified by column chromatography to yield 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%) upon rotary evaporation.

[0189] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine

[0190] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C. under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and the reaction mixture was stirred. After 10 minutes the reaction was warmed to room temperature and stirred for 2 h. while the progress of the reaction was monitored by TLC (5% MeOH in CH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and the product was extracted with EtOAc (2×20 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. This entire procedure was repeated with the resulting residue, with the exception that formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolution of the residue in the PPTS/MeOH solution. After the extraction and evaporation, the residue was purified by flash column chromatography and (eluted with 5% MeOH in CH₂Cl₂) to afford 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%) upon rotary evaporation.

[0191] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0192] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol). The reaction was stirred at room temperature for 24 hrs and monitored by TLC (5% MeOH in CH₂Cl₂). The solvent was removed under vacuum and the residue purified by flash column chromatography (eluted with 10% MeOH in CH₂Cl₂) to afford 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%) upon rotary evaporation of the solvent.

[0193] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0194] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P₂O₅ under high vacuum overnight at 40° C., co-evaporated with anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to the pyridine solution and the reaction mixture was stirred at room temperature until all of the starting material had reacted. Pyridine was removed under vacuum and the residue was purified by column chromatography (eluted with 10% MeOH in CH₂Cl₂ containing a few drops of pyridine) to yield 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%) upon rotary evaporation.

[0195] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0196] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried over P₂O₅ under high vacuum overnight at 40° C. This was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 h under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, then the residue was dissolved in EtOAc (70 mL) and washed with 5% aqueous NaHCO₃ (40 mL). The EtOAc layer was dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue obtained was purified by column chromatography (EtOAc as eluent) to afford 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%) upon rotary evaporation.

[0197] 2′-(Aminooxyethoxy) Nucleoside Amidites

[0198] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as 2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.

[0199] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0200] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may be phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0201] 2′-Dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

[0202] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or 2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly.

[0203] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine

[0204] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) was slowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. (Caution: Hydrogen gas evolves as the solid dissolves). O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) were added and the bomb was sealed, placed in an oil bath and heated to 155° C. for 26 h. then cooled to room temperature. The crude solution was concentrated, the residue was diluted with water (200 mL) and extracted with hexanes (200 mL). The product was extracted from the aqueous layer with EtOAc (3×200 mL) and the combined organic layers were washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (eluted with 5:100:2 MeOH/CH₂Cl₂/TEA) as the eluent. The appropriate fractions were combined and evaporated to afford the product as a white solid.

[0205] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl Uridine

[0206] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino- ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. The reaction mixture was poured into water (200 mL) and extracted with CH₂Cl₂ (2×200 mL). The combined CH₂C1₂ layers were washed with saturated NaHCO₃ solution, followed by saturated NaCl solution, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography (eluted with 5:100:1 MeOH/CH₂Cl₂/TEA) to afford the product.

[0207] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl Uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0208] Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) were added to a solution of 5′-O-dimethoxytrityl-2′-O-[2(2-N,N- dimethylaminoethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere of argon. The reaction mixture was stirred overnight and the solvent evaporated. The resulting residue was purified by silica gel column chromatography with EtOAc as the eluent to afford the title compound.

Example 2 Oligonucleotide Synthesis

[0209] Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0210] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with>3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0211] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference. 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0212] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No., 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0213] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0214] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0215] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0216] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3 Oligonucleoside Synthesis

[0217] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0218] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0219] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

[0220] Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.

Example 5 Synthesis of Chimeric Oligonucleotides

[0221] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[0222] [2′-O-Me]—[2′-deoxy]—[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides

[0223] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH_(4OH) for) 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[0224] [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0225] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[0226] [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxy Phosphorothioate]—[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[0227] [2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxy phosphorothioate]—[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3, H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0228] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

[0229] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with>3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32+/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

[0230] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3, H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta- cyanoethyldiisopropyl phosphoramidites.

[0231] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8 Oligonucleotide Analysis—96-Well Plate Format

[0232] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

[0233] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

[0234] T-24 Cells:

[0235] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, CA), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0236] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0237] A549 Cells:

[0238] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0239] NHDF Cells:

[0240] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0241] HEK Cells:

[0242] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0243] b.END Cells:

[0244] The mouse brain endothelial cell line b.END was obtained from Dr. Werner Risau at the Max Plank Instititute (Bad Nauheim, Germany). b.END cells were routinely cultured in DMEM, high glucose (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 3000 cells/well for use in RT-PCR analysis.

[0245] For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0246] Treatment with Antisense Compounds:

[0247] When cells reached 70% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTINTM (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0248] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (21-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of PPAR-delta Expression

[0249] Antisense modulation of PPAR-delta expression can be assayed in a variety of ways known in the art. For example, PPAR-delta mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1- 4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0250] Protein levels of PPAR-delta can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to PPAR-delta can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997). Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997).

[0251] Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998). Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997). Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991).

Example 11 Poly(A)+mRNA Isolation

[0252] Poly(A)+mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+mRNA isolation are taught in, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993). Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 AL cold PBS. 60 jL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0253] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

[0254] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY ₉₆™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 170 μL water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0255] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13 Real-time Quantitative PCR Analysis of PPAR-delta mRNA Levels

[0256] Quantitation of PPAR-delta mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0257] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0258] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer (—MgCl2), 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension) Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0259] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480nm and emission at 520 nm.

[0260] Probes and primers to human PPAR-delta were designed to hybridize to a human PPAR-delta sequence, using published sequence information (the complement of residues 66001 -170245 of GenBank accession number AL022721.1, incorporated herein as SEQ ID NO:4). For human PPAR-delta the PCR primers were:

[0261] forward primer: ACCCTGATGCCCAGTACCTCTT (SEQ ID NO: 5) reverse primer: GTCTCGGTTTCGGTCTTCTTGAT (SEQ ID NO: 6) and the PCR probe was: FAM-ACCTGCGGCAACTGGTCACCGA-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were:

[0262] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

[0263] Probes and primers to mouse PPAR-delta were designed to hybridize to a mouse PPAR-delta sequence, using published sequence information (a partial genomic sequence was assembled from GenBank accession number AC068495.7, and is incorporated herein as SEQ ID NO:11). For mouse PPAR-delta the PCR primers were:

[0264] forward primer: GTCATCCACGACATCGAGACA (SEQ ID NO:12) reverse primer: GCCCGTTCACCAGCTGTTT (SEQ ID NO: 13) and the PCR probe was: FAM-TGTGGCAGGCAGAGAAGGGCC-TAMRA (SEQ ID NO: 14) where FAM is the fluorescent reporter dye and TAMRA is the quencher dye. For mouse GAPDH the PCR primers were:

[0265] forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO:15) reverse primer: GGGTCTCGCTCCTGGAAGAT(SEQ ID NO:16) and the PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 17) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14 Northern Blot Analysis of PPAR-delta mRNA Levels

[0266] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0267] To detect human PPAR-delta, a human PPAR-delta specific probe was prepared by PCR using the forward primer ACCCTGATGCCCAGTACCTCTT (SEQ ID NO: 5) and the reverse primer GTCTCGGTTTCGGTCTTCTTGAT (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0268] To detect mouse PPAR-delta, a mouse PPAR-delta specific probe was prepared by PCR using the forward primer GTCATCCACGACATCGAGACA (SEQ ID NO: 12) and the reverse primer GCCCGTTCACCAGCTGTTT (SEQ ID NO: 13). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0269] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15 Antisense Inhibition of Human PPAR-delta Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy gap

[0270] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human PPAR-delta RNA, using published sequences (the complement of residues 66001 -170245 of GenBank accession number AL022721.1, incorporated herein as SEQ ID NO: 4; and GenBank accession number NM_(—)006238.1, incorporated herein as SEQ ID NO: 18). The oligonucleotides are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human PPAR-delta mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which A549 cells were treated with the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human PPAR-delta mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap CONTROL TARGET TARGET SEQ SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB ID NO NO 136862 Intron 4 2328 ccagggcagcagttgtaaga 70 19 1 136863 Intron 4 8606 tctgggtgctccagtattgg 77 20 1 136864 Intron 4 13487 tactccctcccttttgcagt 63 21 1 136865 Intron 4 15757 caagtagctgggattacagg 96 22 1 136866 Intron 4 31024 caatatgcttctattaccag 94 23 1 136867 Intron 4 40215 tcctacaacatctcagcctg 84 24 1 136868 Intron 4 55968 tgctaattgtttacacaata 84 25 1 136869 Intron 4 78810 agccctctgtgctcctggtc 94 26 1 136870 Intron 4 89685 tcagtttcaccatctttgat 93 27 1 136871 Intron 4 92621 gcagcaggcacgcgatagct 90 28 1 136872 5′ UTR 18 47 ctgtacaacactgtcccggc 90 29 1 136873 5′ UTR 18 67 tcacgtgcatgcccaaaaca 91 30 1 136874 5′ UTR 18 94 tggtgagcagaagccactgt 89 31 1 136875 5′ UTR 18 98 ctgttggtgagcagaagcca 91 32 1 136876 5′ UTR 18 101 catctgttggtgagcagaag 90 33 1 136877 5′ UTR 18 121 ctcgttggtgcatctgtctt 89 34 1 136878 5′ UTR 18 132 cattccagaccctcgttggt 66 35 1 136879 5′ UTR 18 150 ttccagaccactccagacca 79 36 1 136880 5′ UTR 18 225 ccatcagccttgaagcagtc 66 37 1 136881 5′ UTR 18 228 ttcccatcagccttgaagca 66 38 1 136882 5′ UTR 18 259 gtctgaacgcagatggacct 87 39 1 136883 Start 18 330 tggctgctccatggctgatc 82 40 1 Codon 136884 Coding 18 458 cgggagaggtctgtgtagct 78 41 1 136885 Coding 18 507 gtcacagcccatctgcagtt 85 42 1 136886 Coding 18 539 cactccatgttgaggctgcc 87 43 1 136887 Coding 18 545 acccggcactccatgttgag 90 44 1 136888 Coding 18 835 ccacctgtgggttgtactgg 89 45 1 136889 Coding 18 853 agaaggccttcaggtcggcc 83 46 1 136890 Coding 18 859 gcttggagaaggccttcagg 89 47 1 136891 Coding 18 869 ttgtagatgtgcttggagaa 83 48 1 136892 Coding 18 875 taggcattgtagatgtgctt 91 49 1 136893 Coding 18 880 tcaggtaggcattgtagatg 91 50 1 136894 Coding 18 886 agtttttcaggtaggcattg 90 51 1 136895 Coding 18 911 cgggccttctttttggtcat 90 52 1 136896 Coding 18 1144 tgaggaagaggctgctgaag 90 53 1 136897 Coding 18 1151 tggtcgttgaggaagaggct 90 54 1 136898 Coding 18 1181 tcgtgcacgccatacttgag 90 55 1 136899 Coding 18 1187 atggcctcgtgcacgccata 86 56 1 136900 Coding 18 1239 gttggctaccagcagcccgt 92 57 1 136901 Coding 18 1309 taggctcaatgatatcactg 98 58 1 136902 Coding 18 1394 ccacacagaatgatggccgc 93 59 1 136903 Coding 18 1400 cggtctccacacagaatgat 90 60 1 136904 Coding 18 1406 cctggccggtctccacacag 85 61 1 136905 Coding 18 1412 atgaggcctggccggtctcc 89 62 1 136906 Coding 18 1418 acgttcatgaggcctggccg 85 63 1 136907 Coding 18 1528 ccatcttctgcagcagcttg 94 64 1 136908 Coding 18 1575 ccgctgcatcatctgggcgt 93 65 1 136909 Stop 18 1653 gtgccgccgttagtacatgt 84 66 1 Codon 136910 3′ UTR 18 1736 caggaagagagctggtcaat 79 67 1 136911 3′ UTR 18 1737 acaggaagagagctggtcaa 67 68 1 136912 3′ UTR 18 1932 tcctgttctatgctgctggt 93 69 1 136913 3′ UTR 18 1951 aggtgtgcaaaagcagaggt 91 70 1 136914 3′ UTR 18 2056 ctcaagtcttttgctctgaa 95 71 1 136915 3′ UTR 18 2073 agtgtttctttggatggctc 91 72 1 136916 3′ UTR 18 2086 gcccagagagcttagtgttt 93 73 1 136917 3′ UTR 18 2167 actgtcctttgcagcaggga 85 74 1 136918 3′ UTR 18 2315 aaaccagtgtgaagatggaa 94 75 1 136919 3′ UTR 18 2334 tcagcaacattggcctggca 90 76 1 136926 3′ UTR 18 2337 ccatcagcaacattggcctg 92 77 1 136921 3′ UTR 18 2339 ggccatcagcaacattggcc 91 78 1 136922 3′ UTR 18 2393 tgcatggtgcctgcaggtaa 77 79 1 136923 3′ UTR 18 2442 gcaggcccctctctctgggt 91 80 1 136924 3′ UTR 18 2499 aggacctgcaggacccctgc 89 81 1 136925 3′ UTR 18 2536 gaagctcccactgggcgagg 90 82 1 136926 3′ UTR 18 2542 tcccgggaagctcccactgg 84 83 1 136927 3′ UTR 18 2565 tcagaatgaacaggctcagt 94 84 1 136928 3′ UTR 18 2579 gggacaaatggacatcagaa 86 85 1 136929 3′ UTR 18 2588 agagctattgggacaaatgg 94 86 1 136930 3′ UTR 18 2598 ggagggcagtagagctattg 87 87 1 136931 3′ UTR 18 2660 ggacactagaggctgtgcag 91 88 1 136932 3′ UTR 18 2745 gccctgctctgaggtgagcg 87 89 1 136933 3′ UTR 18 2783 ctgcgccgctcagacatggc 89 90 1 136934 3′ UTR 18 2869 aggaagcctgggcttgaacc 83 91 1 136935 3′ UTR 18 2926 acttccacccagagtcacat 86 92 1 136936 3′ UTR 18 2986 gagcgggaagaatcctgcca 94 93 1 136937 3′ UTR 18 3039 tcatagccttggctgaaaga 73 94 1 136938 3′ UTR 18 3182 gctcctagcaaaaatataca 86 95 1 136939 3′ UTR 18 3229 tcgtcagtctgtgtacacta 94 96 1

[0271] As shown in Table 1, SEQ ID NOs 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 95 and 96 demonstrated at least 80% inhibition of human PPAR-delta expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “preferred target regions” and are therefore preferred sites for targeting by compounds of the present invention. These preferred target regions are shown in Table 3. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number of the corresponding target nucleic acid. Also shown in Table 3 is the species in which each of the preferred target regions was found.

Example 16 Antisense Inhibition of Mouse PPAR-delta Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy gap

[0272] In accordance with the present invention, a second series of oligonucleotides were designed to target different regions of the mouse PPAR-delta RNA, using published sequences (a partial genomic sequence assembled from GenBank accession number AC068495.7, incorporated herein as SEQ ID NO:1l; GenBank accession number L28116.1, incorporated herein as SEQ ID NO: 97; GenBank accession number AW321428.1, incorporated herein as SEQ ID NO: 98; and GenBank accession number U10375.1, incorporated herein as SEQ ID NO: 99). The oligonucleotides are shown in Table 2. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The intercleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residue are 5-methylcytidines. The compounds were analyzed for their effect on mouse PPAR-delta mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which b.END cells were treated with the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”. TABLE 2 Inhibition of mouse PPAR-delta mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET CONTROL SEQ ID TARGET % SEQ SEQ ID ISIS # REGION NO SITE SEQUENCE INHIB ID NO NO 221071 5′ UTR 97 4 tggtcatagctctgccacca 78 100 1 221072 5′ UTR 97 36 ctgacccccacttggcgtgg 92 101 1 221073 Start 97 54 cctgtggctgttccatgact 83 102 1 Codon 221074 3′ UTR 97 1401 tgggcccagcagagcctgag 75 103 1 221075 3′ UTR 97 1418 tctgaacagtccgtggctgg 79 104 1 221076 3′ UTR 97 1439 gccagtgcctgtggctggtc 82 105 1 221077 3′ UTR 97 1467 gttgtgagtaggctctagct 77 106 1 221078 3′ UTR 97 1482 ccacgtgtctggagtgttgt 80 107 1 221079 5′ UTR 98 66 tgcacagcactgtcccggcc 87 108 1 221080 5′ UTR 98 127 tgtcttcatctgtcagtgag 76 109 1 221081 5′ UTR 98 157 agcgcagatggactgccttt 71 110 1 221082 5′ UTR 98 171 accatctgggtctgagcgca 76 111 1 221083 5′ UTR 98 420 tctcccagggcccaaaatca 0 112 1 221084 5′ UTR 98 465 gtccctgggtctgctttgca 53 113 1 221085 Start 99 1 tcctcctgtggctgttccat 84 114 1 Codon 221086 Coding 99 25 tcctcttcccgggcctcagg 84 115 1 221087 Coding 99 41 ggccacttcctctttctcct 95 116 1 221088 Coding 99 81 gttctggtcccccattgagc 78 117 1 221089 Coding 99 117 gggagaggtctgcacagctg 68 118 1 221090 Coding 99 125 ggaattctgggagaggtctg 62 119 1 221091 Coding 99 149 ctggtccagcagggaggaag 69 120 1 221092 Coding 99 154 tgcagctggtccagcaggga 87 121 1 221093 Coding 99 159 ccatctgcagctggtccagc 81 122 1 221094 Coding 99 164 acagcccatctgcagctggt 3 123 1 221095 Coding 99 169 ccatcacagcccatctgcag 86 124 1 221096 Coding 99 175 gaggccccatcacagcccat 89 125 1 221097 Coding 99 202 cgacattccatgttgaggct 62 126 1 221098 Coding 99 293 cttcatgcggattgtccggc 85 127 1 221099 Coding 99 307 ttctcatactcgagcttcat 87 128 1 221100 Coding 99 481 tgctggcacccctcgctggc 85 129 1 221101 Coding 99 506 cttcaggtcggccagctggg 77 130 1 221102 Coding 99 512 gaaggccttcaggtcggcca 40 131 1 221103 Coding 99 570 gggccttctttttggtcatg 58 132 1 221104 Coding 99 577 atgctccgggccttcttttt 58 133 1 221105 Coding 99 582 tgaggatgctccgggccttc 78 134 1 221106 Coding 99 614 gacaaagggtgcgttgtggc 60 135 1 221107 Coding 99 655 aggcccttctctgcctgcca 88 136 1 221108 Coding 99 702 tgatctcgttgtagggcggc 68 137 1 221109 Coding 99 730 gactggcagcggtagaacac 0 138 1 221110 Coding 99 774 tcttggcgaactcggtgagc 77 139 1 221111 Coding 99 779 gatgttcttggcgaactcgg 84 140 1 221112 Coding 99 799 aagaggctgctgaagttggg 55 141 1 221113 Coding 99 843 cctcgtgcacgccatacttg 92 142 1 221114 Coding 99 848 gatggcctcgtgcacgccat 75 143 1 221115 Coding 99 859 agcatggcaaagatggcctc 58 144 1 221116 Coding 99 864 aggccagcatggcaaagatg 57 145 1 221117 Coding 99 904 ctgccgttggccaccagcag 66 146 1 221118 Coding 99 909 agccactgccgttggccacc 79 147 1 221119 Coding 99 914 gacgaagccactgccgttgg 74 148 1 221120 Coding 99 919 tgggtgacgaagccactgcc 74 149 1 221121 Coding 99 931 cgcaagaactcgtgggtgac 78 150 1 221122 Coding 99 945 gcttgcggagacttcgcaag 54 151 1 221123 Coding 99 956 gtcactgaagggcttgcgga 76 152 1 221124 Coding 99 965 ctcaatgatgtcactgaagg 83 153 1 221125 Coding 99 977 ctcgaacttgggctcaatga 84 154 1 221126 Coding 99 1048 agaatgatggccgcgatgaa 47 155 1 221127 Coding 99 1061 ccggtctccacacagaatga 74 156 1 221128 Coding 99 1108 atggtgtcctggatggcttc 80 157 1 221129 Coding 99 1134 gcagatggaattctagagcc 77 158 1 221130 Coding 99 1139 gacctgcagatggaattcta 0 159 1 221131 Coding 99 1144 tggttgacctgcagatggaa 0 160 1 221132 Coding 99 1154 gctgtcagggtggttgacct 78 161 1 221133 Coding 99 1168 gggaagaggtactggctgtc 66 162 1 221134 Coding 99 1226 catctgggcatgctcagtga 0 163 1 221135 Coding 99 1258 gtctcactctccgtcttctt 88 164 1 221136 Coding 99 1263 gcaaggtctcactctccgtc 80 165 1 221137 Coding 99 1268 gtgcagcaaggtctcactct 87 166 1 221138 Intron 11 1954 ccaggatgcactggcccaag 89 167 1 221139 Exon: 11 5752 tgggagaggtctgtgaagac 54 168 1 Intron Junction 221140 Exon: 11 5911 cagtccatctgtaccttgca 84 169 1 Intron Junction 221141 Intron 11 6185 aaagatcctcttaagaccca 73 170 1 221142 Intron 11 8440 tgaccagggcccatgcctga 81 171 1 221143 Exon: 11 8661 aagcggatagctgcataggg 37 172 1 Intron Junction 221144 Exon: 11 8864 tgacactcactgcgttgtgg 0 173 1 Intron Junction 221145 Intron: 11 8968 tgacaaagggctgaaaacca 43 174 1 Exon Junction

[0273] As shown in Table 2, SEQ ID NOs 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 124, 125, 126, 127, 128, 129, 130, 134, 135, 136, 137, 139, 140, 142, 143, 146, 147, 148, 149, 150, 152, 153, 154, 156, 157, 158, 161, 162, 164, 165, 166, 167, 169, 170 and 171 demonstrated at least 60% inhibition of mouse PPAR-delta expression in this experiment and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “preferred target regions” and are therefore preferred sites for targeting by compounds of the present invention. These preferred target regions are shown in Table 3. The sequences represent the reverse complement of the preferred antisense compounds shown in Tables 1 and 2. “Target site” indicates the first (5′-most) nucleotide number of the corresponding target nucleic acid. Also shown Table 3 is the species in which each of the preferred target regions was found. TABLE 3 Sequence and position of preferred target regions identified in PPAR-delta. TARGET SEQ ID TARGET REV COMP ACTIVE SEQ ID SITE ID NO SITE SEQUENCE OF SEQ ID IN NO 49985 4 15757 cctgtaatcccagctacttg 22 H. sapiens 175 49986 4 31024 ctggtaatagaagcatattg 23 H. sapiens 176 49987 4 40215 caggctgagatgttgtagga 24 H. sapiens 177 49988 4 55968 tattgtgtaaacaattagca 25 H. sapiens 178 49989 4 78810 gaccaggagcacagagggct 26 H. sapiens 179 49990 4 89685 atcaaagatggtgaaactga 27 H. sapiens 180 49991 4 92621 agctatcgcgtgcctgctgc 28 H. sapiens 181 49992 18 47 gccgggacagtgttgtacag 29 H. sapiens 182 49993 18 67 tgttttgggcatgcacgtga 30 H. sapiens 183 49994 18 94 acagtggcttctgctcacca 31 H. sapiens 184 49995 18 98 tggcttctgctcaccaacag 32 H. sapiens 185 49996 18 101 cttctgctcaccaacagatg 33 H. sapiens 186 49997 18 121 aagacagatgcaccaacgag 34 H. sapiens 187 50002 18 259 aggtccatctgcgttcagac 39 H. sapiens 188 50003 18 330 gatcagccatggagcagcca 40 H. sapiens 189 50005 18 507 aactgcagatgggctgtgac 42 H. sapiens 190 50006 18 539 ggcagcctcaacatggagtg 43 H. sapiens 191 50007 18 545 ctcaacatggagtgccgggt 44 H. sapiens 192 50008 18 835 ccagtacaacccacaggtgg 45 H. sapiens 193 50009 18 853 ggccgacctgaaggccttct 46 H. sapiens 194 50010 18 859 cctgaaggccttctccaagc 47 H. sapiens 195 50011 18 869 ttctccaagcacatctacaa 48 H. sapiens 196 50012 18 875 aagcacatctacaatgccta 49 H. sapiens 197 50013 18 880 catctacaatgcctacctga 50 H. sapiens 198 50014 18 886 caatgcctacctgaaaaact 51 H. sapiens 199 50015 18 911 atgaccaaaaagaaggcccg 52 H. sapiens 200 50016 18 1144 cttcagcagcctcttcctca 53 H. sapiens 201 50017 18 1151 agcctcttcctcaacgacca 54 H. sapiens 202 50018 18 1181 ctcaagtatggcgtgcacga 55 H. sapiens 203 50019 18 1187 tatggcgtgcacgaggccat 56 H. sapiens 204 50020 18 1239 acgggctgctggtagccaac 57 H. sapiens 205 50021 18 1309 cagtgatatcattgagccta 58 H. sapiens 206 50022 18 1394 gcggccatcattctgtgtgg 59 H. sapiens 207 50023 18 1400 atcattctgtgtggagaccg 60 H. sapiens 208 50024 18 1406 ctgtgtggagaccggccagg 61 H. sapiens 209 50025 18 1412 ggagaccggccaggcctcat 62 H. sapiens 210 50026 18 1418 cggccaggcctcatgaacgt 63 H. sapiens 211 50027 18 1528 caagctgctgcagaagatgg 64 H. sapiens 212 50028 18 1575 acgcccagatgatgcagcgg 65 H. sapiens 213 50029 18 1653 acatgtactaacggcggcac 66 H. sapiens 214 50032 18 1932 accagcagcatagaacagga 69 H. sapiens 215 50033 18 1951 acctctgcttttgcacacct 70 H. sapiens 216 50034 18 2056 ttcagagcaaaagacttgag 71 H. sapiens 217 50035 18 2073 gagccatccaaagaaacact 72 H. sapiens 218 50036 18 2086 aaacactaagctctctgggc 73 H. sapiens 219 50037 18 2167 tccctgctgcaaaggacagt 74 H. sapiens 220 50038 18 2315 ttccatcttcacactggttt 75 H. sapiens 221 50039 18 2334 tgccaggccaatgttgctga 76 H. sapiens 222 50040 18 2337 caggccaatgttgctgatgg 77 H. sapiens 223 50041 18 2339 ggccaatgttgctgatggcc 78 H. sapiens 224 50043 18 2442 acccagagagaggggcctgc 80 H. sapiens 225 50044 18 2499 gcaggggtcctgcaggtcct 81 H. sapiens 226 50045 18 2536 cctcgcccagtgggagcttc 82 H. sapiens 227 50046 18 2542 ccagtgggagcttcccggga 83 H. sapiens 228 50047 18 2565 actgagcctgttcattctga 84 H. sapiens 229 50048 18 2579 ttctgatgtccatttgtccc 85 H. sapiens 230 50049 18 2588 ccatttgtcccaatagctct 86 H. sapiens 231 50050 18 2598 caatagctctactgccctcc 87 H. sapiens 232 50051 18 2660 ctgcacagcctctagtgtcc 88 H. sapiens 233 50052 18 2745 cgctcacctcagagcagggc 89 H. sapiens 234 50053 18 2783 gccatgtctgagcggcgcag 90 H. sapiens 235 50054 18 2869 ggttcaagcccaggcttcct 91 H. sapiens 236 50055 18 2926 atgtgactctgggtggaagt 92 H. sapiens 237 50056 18 2986 tggcaggattcttcccgctc 93 H. sapiens 238 50058 18 3182 tgtatatttttgctaggagc 95 H. sapiens 239 50059 18 3229 tagtgtacacagactgacga 96 H. sapiens 240 137725 97 4 tggtggcagagctatgacca 100 M. musculus 241 137726 97 36 ccacgccaagtgggggtcag 101 M. musculus 242 137727 97 54 agtcatggaacagccacagg 102 M. musculus 243 137728 97 1401 ctcaggctctgctgggccca 103 M. musculus 244 137729 97 1418 ccagccacggactgttcaga 104 M. musculus 245 137730 97 1439 gaccagccacaggcactggc 105 M. musculus 246 137731 97 1467 agctagagcctactcacaac 106 M. musculus 247 137732 97 1482 acaacactccagacacgtgg 107 M. musculus 248 137733 98 66 ggccgggacagtgctgtgca 108 M. musculus 249 137734 98 127 ctcactgacagatgaagaca 109 M. musculus 250 137735 98 157 aaaggcagtccatctgcgct 110 M. musculus 251 137736 98 171 tgcgctcagacccagatggt 111 M. musculus 252 137739 99 1 atggaacagccacaggagga 114 M. musculus 253 137740 99 25 cctgaggcccgggaagagga 115 M. musculus 254 137741 99 41 aggagaaagaggaagtggcc 116 M. musculus 255 137742 99 81 gctcaatgggggaccagaac 117 M. musculus 256 137743 99 117 cagctgtgcagacctctccc 118 M. musculus 257 137744 99 125 cagacctctcccagaattcc 119 M. musculus 258 137745 99 149 cttcctccctgctggaccag 120 M. musculus 259 137746 99 154 tccctgctggaccagctgca 121 M. musculus 260 137747 99 159 gctggaccagctgcagatgg 122 M. musculus 261 137749 99 169 ctgcagatgggctgtgatgg 124 M. musculus 262 137750 99 175 atgggctgtgatggggcctc 125 M. musculus 263 137751 99 202 agcctcaacatggaatgtcg 126 M. musculus 264 137752 99 293 gccggacaatccgcatgaag 127 M. musculus 265 137753 99 307 atgaagctcgagtatgagaa 128 M. musculus 266 137754 99 481 gccagcgaggggtgccagca 129 M. musculus 267 137755 99 506 cccagctggccgacctgaag 130 M. musculus 268 137759 99 582 gaaggcccggagcatcctca 134 M. musculus 269 137760 99 614 gccacaacgcaccctttgtc 135 M. musculus 270 137761 99 655 tggcaggcagagaagggcct 136 M. musculus 271 137762 99 702 gccgccctacaacgagatca 137 M. musculus 272 137764 99 774 gctcaccgagttcgccaaga 139 M. musculus 273 137765 99 779 ccgagttcgccaagaacatc 140 M. musculus 274 137767 99 843 caagtatggcgtgcacgagg 142 M. musculus 275 137768 99 848 atggcgtgcacgaggccatc 143 M. musculus 276 137771 99 904 ctgctggtggccaacggcag 146 M. musculus 277 137772 99 909 ggtggccaacggcagtggct 147 M. musculus 278 137773 99 914 ccaacggcagtggcttcgtc 148 M. musculus 279 137774 99 919 ggcagtggcttcgtcaccca 149 M. musculus 280 137775 99 931 gtcacccacgagttcttgcg 150 M. musculus 281 137777 99 956 tccgcaagcccttcagtgac 152 M. musculus 282 137778 99 965 ccttcagtgacatcattgag 153 M. musculus 283 137779 99 977 tcattgagcccaagttcgag 154 M. musculus 284 137781 99 1061 tcattctgtgtggagaccgg 156 M. musculus 285 137782 99 1108 gaagccatccaggacaccat 157 M. musculus 286 137783 99 1134 ggctctagaattccatctgc 158 M. musculus 287 137786 99 1154 aggtcaaccaccctgacagc 161 M. musculus 288 137787 99 1168 gacagccagtacctcttccc 162 M. musculus 289 137789 99 1258 aagaagacggagagtgagac 164 M. musculus 290 137790 99 1263 gacggagagtgagaccttgc 165 M. musculus 291 137791 99 1268 agagtgagaccttgctgcac 166 M. musculus 292 137792 11 1954 cttgggccagtgcatcctgg 167 M. musculus 293 137794 11 5911 tgcaaggtacagatggactg 169 M. musculus 294 137795 11 6185 tgggtcttaagaggatcttt 170 M. musculus 295 137796 11 8440 tcaggcatgggccctggtca 171 M. musculus 296

[0274] As these “preferred target regions” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these sites and consequently inhibit the expression of PPAR-delta.

Example 17 Western Blot Analysis of PPAR-delta Protein Levels

[0275] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to PPAR-delta is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 296 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial Sequence Antisense Oligonucleotide 3 atgcattctg cccccaagga 20 4 104245 DNA Homo sapiens 4 gatcatgagg tcaggagttc aagaccagcc tggccaacag gatgaaaccc cgtctctact 60 aaaaatacaa aaattagctg ggcatggtgg cacgtgcctg tagtcccagc tactcgggag 120 gctgaggcag aagaattgct tgaacccagg agacagaggt tgcagtgagc cgaaatcacg 180 ccactgcact ccagcctggg tgacagagca agactctgtc tcaaaaaaaa aaaaaaaaaa 240 aaaattaggc tgggtagggc caggcgcggt ggctcatacc tgtaatccca gcactttggg 300 aggccaaaat gggtggatca caaggtcagg agttcgagac tagcctggcc aacatggtga 360 aaccccgtct ctactaaaaa tacaaaaatt agccaggcat ggtggcgtgt gcctgtagtc 420 ccagctactc aggaggctga ggcaggagaa tcgcttgaac ccgggaggca gaggctgcag 480 tgagctgaga tcacgccact gcactccagc ctgggcaaca gagtgagact ccgtctcaaa 540 aaaaaaaaaa ttagactggg cgtggtggtg tgcacctgta ttcccaatta ctccggaggc 600 tgaggtggga ggattgcttg agtccaggag gcagaggttg tagtgagcca tgatagtgcc 660 acttcactcc agcttgagtg acagagtgag actctgtttc caaaaaataa aaaaatttaa 720 aaaattgatt ggctaaaaaa tgcttcatct tagataaagc ttggaattac tatcttaaaa 780 aatattcaag gggcggccag gcgcggttgg ctcacgcctg caatcctagc actttgggag 840 gccgaggcgg gcggatcaca aggtcaggag atcgagacca tcctggctaa cacggtgaaa 900 ccccgtctct actaaaaata caaaaaaatt agccgggtgt ggtggtgggc gcctatagtc 960 ccagctactc gggaggctga ggcaggagaa tggggtgaac ccaggaggca gagcttgcag 1020 tgagccaaga tcgcaccact gcactccagc ctgggtgaca gagtgagact ccgtctcaag 1080 aaaataaata aataaaataa aataaaaaat atgcaaggga ctgtatggtt cttatatatt 1140 ctcacaggat tggaagggca attttgctat ggagaaccca cgagtagaaa ggtggagtga 1200 gacctggtta tagagattct tggaagctac gcagagttta gatttgaggt ggcaagccaa 1260 cctctgagcc tcagtttcct cacttgtaaa tggggtccat tccaggcttt taaatattct 1320 atggtgggac atagggatgg atcgacagtg gaggcatgat atagtgatat aatgaaagga 1380 agatcactct gacacctgaa tgcgagccat gatgggactg gcatgagaag ccatcaacct 1440 tgggggacat ttctaggtgg aaggagaatg tattttctag tcttttaatt ccagggtgag 1500 aaaataactc ctgtgtcctt tccatcaccc tggacaaaga aaagtgtgtc ctgtctctga 1560 tgccttcaca gatacacata aaccctaggg gccatatgga atgcacagca tttgtggtcc 1620 cacgtcttct ctttttctgt ggcttgtatg tgaggatgga tggggaagtg gggctggagt 1680 acaagaggcg tgttagcaac tattgccagg agtccacctg gcccaagggg tgcctactga 1740 gtcgcctctc tagttcctgc agcagagggg aaggagaggg cgatgcctgg caggctgatt 1800 tcagaagact gaggaggccc ccacctgctg gctagaggct gaaatggcta tggagaatca 1860 aattcagagg caatccagcc ttcccactct gcactgtaag ttgttcaggt acacaataat 1920 caggcatcag agcaggtttt ggtagctgaa atccctgtct ggtctggaac gggctgtaga 1980 tgcttggcag gaatcaactt ttcccactct gagctgtccc ccagggcaaa tctagtcaaa 2040 atgaagattc aaggaagcta ttttagtcta ggtttgtgat tcaagtaacg gaaactgact 2100 gatttaagct gacttaactg actttccaga aaagaaattt attagtagaa tataagggag 2160 taccttaaat aattgggagg tcgagtgagc tgtcttggag gctgtgtagt cagaaacagt 2220 acccaaatca agccacacaa cagttcttgt gaacacactc cagctgccac ctgtgggtac 2280 agaagctgct gcttccttgg ctaatcttac caaatgctgg atgctgctct tacaactgct 2340 gccctggctg tctctggaaa ttgggtggaa ctgctgctgc cactcacctg aatcaattat 2400 gtgtgcttcc tggttcatct ttccaccttc caatacatct gattggtgtg ccttggtcat 2460 gtgactgtgt cctagctgca caggaggctg gaagaaaatg cctgtagttc cagctactct 2520 ggaggctgag gtgggaagat tgctgcttca gcccaggaag cagagattgc cgagatcact 2580 ccactgcact ccaacccagg caacagagca agaccctgtc tcaaaaaaaa agaaagagag 2640 agagagagag agaaagaggg agaagaaaga aaggagggag ggagagagag aggaaggaag 2700 gaaggaaggg aggaaggaag gaaggaagga aagaaaatat ataggggaat gaagtcaaat 2760 gtcaaacatc agaaaagctc ttacatagaa aataattttg acttagttta cctccaatgg 2820 gtagaactaa gattattggg taagagcatc agaggatata tgttttttct taatataaag 2880 aagatttttg ttgttcttag aaaatagcca ggcaaggtgg ctcacacctg taatcccagc 2940 actttgggag gccaaggcga gtaaatcacc tgaggtcagg agttccagac aagcatggcc 3000 aacatgttga aacctgtctc tactaaaaat acaaaaatta gccagtgtgg tggagggtgc 3060 ctgtaatccc agctactcgg gaggctgagg catgagaatc gcttgaacct gggaggtgga 3120 ggttgcattg tgcctagatc acgccactgc actccagtct gggcgacaca gctagactcc 3180 atctcaaaaa aaaaaagaag aagaagaaga aaataaacaa tgtttctatt atggaaaaaa 3240 agcatgctca gaaaaaaaac atagaaaaac aataacttgc agaaccacaa tccaaagaca 3300 gttactggta acatttttgt gtgtttcctt tcagtttttt tgtttgtttg tttttgttta 3360 ttttggagag agagtcttgc tctgttgtcc aggctggagt gaagtggcac gatcttggct 3420 cactgcagcc tccacctcct gggatcacgc gattctcctg cctgagcctc ctgagtagct 3480 gggactacag gcgcctgcca ctacgcccgg ctaatttttt ttgtattttt agtagagacg 3540 ggtttcacca tgttgatcac gctggtctcg aactcctgat gttaagtgat ccaccctcct 3600 cagcctccca aagtgctggg attataggtt gagtcactgt gcccggaatt tttttttttt 3660 ttttgagaca gggtctcact ctgacgccag gctggagtgc agcagagcaa tctcagctca 3720 ctgcaacctc cacctcccag gttcaagcga tccttgtgcc tcagcctact gagtagctgg 3780 tactataggc acacgccacc atgcccggct cctttcagtt tttttccatg ctaattttta 3840 gttttcaaaa cagtgtgatt tcactccata cgtatcatca tatccttttc cacttaatat 3900 cagattataa catttttcca ctttattatt gtccaaaaga ccaccatgat ggttaaatag 3960 tagataggag agctttacta agtgatacca gtttgcaaac caggaagaga tagtctcaga 4020 catggactga aggtgctctc tattctcttc aaagagggaa agggcaggtt gggttttaag 4080 cctcacaggg tctgtactac acaatagtca tacatattta gcggttttgg gggaaaaact 4140 atacatattt atgaggggag ccaagtacat gtgcaatggg caaacatata tgtaacataa 4200 atcccatgtt cactttgggg caggtttcag cattaaaatg aggtggaatt tggctcttta 4260 catcaaaggt gaactgtaga acacaaagac ggtttgtgtg gagcctctat aaactggctg 4320 aaactggttt aaggtctgca actgcttatc aaaatagaat gtttgtaggc cagtggctca 4380 tgcctgtaat cccagcactt tgggaggcca aggtgggtgg attgcttgtg ctcaggagtt 4440 cgagacaagc ctgggcaaca tggggagctc ccatctcaat tataaaaaat aaaaaatgtt 4500 aaaaaaataa agaaaagggc tgggcgcggt ggctcacacc tgtaatccca gcactttggg 4560 aggctgaggc gggcagatca cctgaggtca ggagtttgag accagcctga ccaacatgga 4620 gaaaccccat ctctactaaa aatacaaaat tagctgggcg tggtggcaaa tgcctgtaat 4680 cccagctact caggaggctg aggcaggaga atcgcttgaa ccctggaggc agaagttgtg 4740 gtgagctgag atcgcgccat tgcactctag cccgggcaac aggagcgaaa ctcggtctca 4800 aaaaacaaaa gaaagaaaga aaagaatgta aggccagtcc tctgtccaat cagagttgta 4860 gtggtctggc ttgtaaatta gctaggcgag gtctgatcat ttgcctgata cctcctgttg 4920 ttgagacagt ttatccagaa tgtggttttt cctatagcca caggaattta gggagttgcc 4980 atgccagctg cgttgaaccg tataattaac ctttgtttcc ttaaccttag gttctatctt 5040 agtgataaag gggtgtgtgt tttggtttct cagaccatat taccagctct ttttaaatat 5100 catttttaat ggctgcataa tattccatca gaaggataca gcatgattta cctaaacatt 5160 tctctgttgt tggacaatta ggttatctcc agttttttgt tggtttaaat aatactgaat 5220 gagcattttt gtgtacaaag ccttttatgt atttaggatt atttcctcaa gcagactatc 5280 caaggtagaa ttatgcgttc taaataataa atatagagat aggtatttat tagatatatt 5340 aaatttcata tattcttaat ataaaaatga agtgaaaaat agaataactt aacagtgctc 5400 cttgaatttc ttttaggaga ggaattgtct ttttgttttt ttgagacagt cttgctctgt 5460 cacccaggct ggagtgcaat ggcacgatct tggttcactg caaactccac cttccaggtt 5520 caagtgattc tcctgcttca gcctcctgag tagctgggat tacaggcatg tgccaccacg 5580 tctggctaat tttttgtact ttttagtaga gacagggttt cactgtgtta gccagggtgg 5640 ttgcaatctc ctgacctcgt gatccaaact gtctctatta ggaatgttaa cttaaaaatc 5700 acaaatttgg gccaggcacg atggctcatg cctataatcc cagcactttg ggaggtcgag 5760 gcgggtggat catgaggcca ggagattgag accatcctgg ctaacacagt gaaaccctgt 5820 ctctactaaa aatacaaaaa attagccggg catggtggcg ggctgtagtc ccagctgctt 5880 gggaggctga ggcaggagaa tggtgtgaac tcaggaggca gagcttgcag tgagctgaga 5940 tcacaccact gcactccagc ctgggtgaca gagcaagact ccatctcaaa aaaaaatcac 6000 aaatttggcc aggcgtggtg gctcacgcct gtaatcccaa cattttggga ggctgaggca 6060 gacagatcac ttgaggtcag gagttccaga ccagcctggc caacatggca aaatcccgtc 6120 tctactaaaa atacaaaaat tagcacacgg ctgaatagga acagttccag tctgcagctc 6180 ccagggtgat caacgcagaa gatgggtgat ttctgcattt ccaactgagg tacctggttc 6240 atctcactgg gactggttgg acagtgggtg cagcctacgg agggcaagcc aaagcagggc 6300 aggacatcac ttcacacagg aagcagaagg ggttggggga tttccctttc ctagccaaag 6360 gaagccgaga cagactgtac ctggaaaaac aggacactcc tgcctaaata ctgtgctttt 6420 ccaatggtct tagtaaacgg cacaccagga gattatatgc cacgcatggc ttggagggtt 6480 ccacgcccac agagccttgc tcactgctag cacagcagtc tgagattgac atgcgaggca 6540 gcagcctggc agcagcctgg cagtgggagg ggcatctgcc attgctgagg cttgagtagg 6600 taaacaaagc agccagggaa gcttgaactg ggcggagtcc actgcagctc agcaaagcct 6660 gctgcctctg ttgactctac ctctaggggc agggcatagc tgaacaaaag taggcagaaa 6720 cttctgcaga cttaaaagtc cctatctgac agctcttaag agcagtggtt ctcccagcat 6780 ggcatttgag ctctgggaac agacagactg cctcctcaag tgggtccctg acccctgtgt 6840 agcctaactg ggagcacctc ccagtagggg tcgactgaca cctcatacag gcgggtgctc 6900 ctctgggatg aagcttccag aggaaggatc aggcagcaat atttgctgtt ctgcaatatt 6960 tgctgttctg cagcctctgc tggtgatacc caggcaaaca gggtctggag tggacctcca 7020 gcaaactcca acagacctgc agctgaggga cctgactgtt agaaggacaa ctaacaaaca 7080 gaaaggaata gcatcaacat taacaaaaat gatatccaca ccaaaacccc atccataggt 7140 caccaacatc aaagaccaaa ggtagataaa accacaaaga tggggagaaa acagagcaga 7200 aaagctgaaa attctaaaaa ccagagtgcc tcttctcttc caaaggattg cagctcctca 7260 ccagcaatgg aaaaaagctg gacgaagaat gactttagtt gacagaagta ggcttcagaa 7320 ggttggtaat gacaaacttc tctgagctaa aggaggatgt tcgaacccat cgcaaggaag 7380 acaaaaacct tgaaaaaaga ttagacaaat ggctaactag aataaacagt gtagagaaga 7440 ccttaaatga cctgatggag ctgaaaacca tggcatgaga actacgtgac acatgcacaa 7500 gcttcagtag ctgattcgat caagtggaag aaagggtatc agtgattgaa gatcaaatta 7560 atgaaataaa gtgagaagag aagtttagag aaaaaagagt aaaaagaaat gaacaaagcc 7620 tccaagaaat atgggactat gtgaaaagac caaatctaca tttgattggt gtacttgaaa 7680 gtgatgggga gaatgaagcc aagttggaaa acactcttca ggatattatc caggagaact 7740 tccccaacct tgcaaggcag gccaacattc aaattcagga aatacagaga acactacaaa 7800 gatactcctc gagaagagca accccaagac acataattgt cagattcacc aaggttgaaa 7860 tgaaggaaaa aatgttaagg gcagccagag agaaaggtca ggttgcccac aaagggaagc 7920 ccatcagact aacagtggat ttctcagcag aaactctaca agccagaaga gagtgggggc 7980 caatattcaa cattcttcaa gaaaagaatt ttcaacccag aatttcatat ccagccaaac 8040 taagcttcat aagtgaaaga gaaataaaat cctttacaga caagcaaatg ctgagatttt 8100 gtcaccacca ggcctgcctt acaagagctc ctggaggaag cactaaacat ggaaaggaac 8160 aaccggtatc agccaccgca aaaacatgcc aaattgtaaa gaccactgtt gctaggaaga 8220 gactgcatca actaatgggc aaaataacca gctaacatca taatgacagg atcaaattca 8280 cacataacaa tattaacctt aaatgtaaat ggggtaaatg cccaattaaa agacacagac 8340 tggcagattg gataaagagt aagacccatc agtgtgctgt attcaggaga cccatctcac 8400 gtgcacagac acacataggc tcaaaataaa gggatggagg aagatctgcc aagcaaatgg 8460 aaagcaaaaa aaagcagggg ttgcaatcct agtctctgat aaaacagact ttaaaccaac 8520 gaagatgaaa agagacaagg ccattacata atggtaaagg gatcaattca acaagaggaa 8580 ctaactatcc taaatatttg tgcacccaat actggagcac ccagattcat aaagcaagtc 8640 cttagagacc taaaaagaga cttagactcc cacacaatag taatgggaga ttttaacacc 8700 ccactgtcaa cattaaacag atcaacgaga cagaaggtta acaaggatat ccaggatttg 8760 aactcagctc tgcaccaagc caacctaata gacatctaca gaactctcca ccacaaatca 8820 acagaatata cattcttctc agcaccacat tgcacttatt ccaaaattga ccacatagtt 8880 ggaagtaaag cactcctcag caaatgtaaa agaccacaac aaactgtctc tcagaccaca 8940 gtgaaatcaa attagaactc aggattaaga aactcagtga aaaccacaca actacatgga 9000 aactgaacaa cctgctcctg aatgactact gggtaaataa cgaaatgaag gcagaaataa 9060 agatgttctt tgaaaccaat gagaacaaag acacaacata ccagaatctc tgggacacat 9120 ttaaagcagt gtgtagacgg aaatttatag cactaaatgc ccacaagaga aagcaggaaa 9180 gatctaaaat cgacacccta acatcacaat taaaagaact agagaagcaa gagcaaacaa 9240 attcaaaagc tagcagaagg caagaaataa ctaagagcaa aacagaaccg aaggagatag 9300 agacacgaaa aacccttcaa aaaatcaatg aatccaggag ctggtgtttt gaaaagatca 9360 acaaaattga tagaccgcta gcaagactaa taaagaagaa aagagagaag aatcaaatag 9420 acgcaataaa aaatgataaa ggggatatca ccaccaatcc cacagatata caaactacca 9480 tcagagaata ctataaacac ctctaagcaa ataaactaga aaatctagaa gaaatggata 9540 aattcctgga cacatacacc ctcccaagaa taaaccagga agatggtgaa tctctgaata 9600 gaccaataac aggctctgaa attgaggcaa taatcaatag cctaccaacc aaaaaaagtc 9660 caggaccaga tggattcaca gccgaattct acgagaggta caaagaggag ctggtaccct 9720 tccttctgaa actattccaa tcaagagaaa aagagggaat cctccctaac tcattttatg 9780 atgccatcat catcctgata ccaaagcctg gcagagacac aacaacaaaa aagagaattt 9840 tagaccaata tgcctgatga acattgatgc gaaaatcctc aataaaatac tgacaaaccg 9900 aatccagcag cacatcaaaa agcttatcca ccatgatcaa gtcggattca tccctgggat 9960 gcaaggctgg ttcaacatac acaaatcaag aaacgtaatc catcacataa acagaaccaa 10020 cgacaaaaac cacatgatta tctcaataga tacagaaaag accttcaaca aaattcaaca 10080 acccttcatg ctaaaaattc tcaataaact aggtattgat gggacgtatc tcaaaataat 10140 aagagctatt tatgacaaac ccacagccaa tatcatactg aatgggcaaa aactggaagc 10200 attccctgtg aaaactggca caagacaggg atgccctctc tcaccactcc tattcaacat 10260 agtgttggaa gttctggcca gggcagtcag gcaagagaaa gaagtaaagg gtattcaatt 10320 aggaaaagag gaagtcaaat tgtccctgtt tgcagatgac atgattgtat atttagaaaa 10380 ccccatcatc tcagcccaaa atctccttaa gctgataagc aagttcagca aagtctcaag 10440 atacaaaatt aatgtgcaaa aatcacaagc attcctatac accaataaca gacaaacaga 10500 gagccaaatc atgagtgaac tcccattcac aattgctaca aagagaataa aatacctagg 10560 aatacaactt acaagggatg tgaaggacct cttcaaggag aactacaaac cactgctctt 10620 tttttttttt tctgagacaa tctcgctctg tcatcaaggc tggagtgcag tggtgcaatc 10680 tcagctcact gcaacctctg cctcccgggt tcaagcgact ctcctgcctc agcctcccaa 10740 gtggctggga ttacaggcgc tcaccaccac gcccagctaa tttttttata tatatatact 10800 ttaagttcta gggtatgtat gcacaatgtg caggtttgtt acataggtat acatgtgcca 10860 agttggtttg ctgcacccat taactcatca tttacgtaag gtatttctcc taatgctatc 10920 cctcccccag ccccccaccc catgacaggc cctggtgtgt gatgttccct gccctgtgtc 10980 caagtgttct cattgttcaa ttcccaccta tgagtgacaa tatgtggtgt ttggttttct 11040 gtctgtgtga tggtttgctc agaataatgg tttccagctt catccatgtg cctgcaaagg 11100 acatgaactc atcatttttt atggctgcac agtattccat ggtgtatatg tgccacattt 11160 tcttagtcca gtctatcatt gatggacatt tgggttggtt ccaagtcttt gctattgtga 11220 atagtgctgc aataaacata cgtgtgcatg tgtctttaca gtagcatgat ttataattct 11280 ttaggtatat acccagtaat gggatcactg ggtcaaatgg tatttctagt tctagatcct 11340 tgaggaatcg ccacactgtc ttccacaatt gttgaactag tttacactcc caccaacagt 11400 gtaaaagcgt tcctgtttct ccacatcctc tccagcatct gttgtttcct gaccttttaa 11460 tgatcgtcat tctaactcgt gtgagatggt atctcattgt ggttttgatt tgcatttctc 11520 tgatgaccag tgatgatgag cattttttca tgtgtctgtt ggctgcataa atgtcttctt 11580 ttgagaagtg tctattcata tcctttgccc actttttgat ggggttgttt ttttcttgta 11640 aatttgttta agttcctcgt agattctgga tattaaccct ttgtcagatg gatagattgc 11700 aaaaattttc tcccattctg taggttgcct gttcactctg atggtagttt cttttgctgt 11760 gcagaagctc ttttggcttt tgttgccatt gcttttggtg ttttagtcat gaagtccttg 11820 ccgatgccta tgtcctgaat ggtattgcct aggctttctt ctagggtttt catggtttta 11880 ggtctaacat ttaagtcttt aatccatctt gaattaattt ttgtgtaagg tataaagaag 11940 aaatccagtt tcagctttct acatattgct agccagtttt cccagcacca tttattaaat 12000 aaggaatcct ttccccattt cttgtttttg tcagatttgt caaagatctg atggttgtag 12060 atgtgtggta ttatttatga ggcctgtgtt ctgttgcatt ggtctatatc tctgttttgg 12120 taccagtacc atgctgtttt ggttactgta gccttgtagt acagtttgaa gtcaggtagt 12180 gtgatgcctc cagctttgtt cttttggctt aggattgtct tggaaatgtg ggcttttttg 12240 gctccatatg aactttaaag tagttttttc caattctgtg aagaaagtca ttggtagctt 12300 gatggggatg gcactgaatc tataaattac cttgggcagt atggccattt tcacgatatt 12360 gattcttcct atccatgagc atggaatgtt cttccatttg tttgtgtcct cctttatttc 12420 attgagcagt ggtttgtagt tctccttgaa gaggtccttc acatcccttg taagttgtat 12480 tcctaggtat tttattctct ttgtagcaat tgtgaatggg agttcactca tgatttggct 12540 ctctgtttgt ctgttattgg tgtataggaa tgcttgtgat ttttgcacat taattttgta 12600 tcttgagact ttgctgaact tgcttatcag cttgaggaga ttttgggctg agatgatggg 12660 gttttctaaa tatacaatca tgtcatctgc aaacagggac aatttgactt cctcttttcc 12720 taattgaata ccctttactt ctttctcttg cctgactgcc ctggccagaa cttccaacac 12780 tatgttgaat aggagtggtg agagagggca tccctgtctt gtgccagttt tcacagggaa 12840 tgcttccagt ttttgcccat tcagtatgat attggctgtg ggtttgtcat aaatagctct 12900 tattattttg agatacgtcc catcaatacc tagtttattg agagttttta gcatgaaggg 12960 ttgttgaatt ttgttgcagg tacttgaaag gaagaggggc tgggacagga gctttatgct 13020 gaacaggttg gctaaacata catattctgg ctaatttttt tgtgtatttt tagtagagat 13080 ggggtttcac catgttagcc aggatggtct cgatctcctg acctcgtgat ctgcctgcct 13140 aggcctccca aagtgctggg attacaggcg tgagccgccg ttcttttttt tttttttttt 13200 ttttaagaga cagggtctca ctatgttgtc caggctggtc tcgaactcct gcgctcaagc 13260 agtctgcccc cctcggcctc tgaaagtgtt ggaattacag gcgtgagcca ccgtgcctgg 13320 cagaaaatat agtttattct ttaggtgtag gcgtgtgtga cttaaccctt acctgacacg 13380 gccttaggtc ctgattataa tttggtatct tattgccata aagagtgcat tctgttagtc 13440 tatgatctct attttaacat tgatgctggt cagatgttgt gtctgaactg caaaagggag 13500 ggagtataac caggcatgtc tgaccccctg acctgtcatg gctggaaact cagtttttaa 13560 ggtttttctg gggtctcctt ggccaagagg gtccattcaa ttagttaggg ggcttaggat 13620 ttgtttttag tttacagggg aaataagcct attaggggta gacagatctg caaagcatga 13680 gtgttggcag gaacttaagc aacaaagaga tacggttaaa atgtcgcttt ctctttctcc 13740 gtagaaagcc agaggaattt gtgttttctc cagagagggt gggacagagg agagaatggt 13800 ggggcaggag ggagagttag tgattttgga ggaggctaga gggtgcaggg ccagttagaa 13860 aacctcagct gggggtgtgg aaaggacttc taaaaacttc tgaggggccc cctccccctc 13920 ccccgctccc tctccccacg gtctccctct ctttccacgg tctccctctc atgcggagcc 13980 gaagctggac tgtactgctg ccatctcggc tcactgcaac ctccctgcct gattctcctg 14040 cctcagcctg ccgagtgcct gcgattgcag gcacgcgtcg ccacgcctga ctggttttgg 14100 tggagacggg gtttcgctgt gttggccggg ccggtctcca gcccctaacc gcgagtgatc 14160 cgccagcctc ggcctcccga ggtgccggga ttgcagacgg agtctcgttc actcagtgct 14220 caatggtgcc caggctggag tgcagtggcg tgatctcggc tcactacaac ctacacctcc 14280 cagccgcctg ccttggcctc ccaaagtgcc gagattgcag cctctgcctg gccgccaccc 14340 cgtatgggaa gtgaggagtg tctctgcctg gccgcccatc gtctgggatg tgaggagccc 14400 ctctgcctgg ctgcccagtc tggaaagtga ggagcgtctc cgcccggccg ccatcccatc 14460 taggaagtga ggagcgcctc ttcccggccg ccatcacatc taggaagtga ggagcgtctc 14520 tgcccggccg cccatcgtct gagatgtggg gagcgcctct gccccgccgc cccatctggg 14580 atatgaggag cgcctctgcc cggcagcgac cccgtctggg aggtgaagag cgtctctgcc 14640 cggccgcccc gtctgagaag tgaggagacc ctctgcctgg caaccacccc gtctgagaag 14700 tgaggagccc ctccgcccgg cagctgcccc gtctgagaag tgaggagcct ctccgcccgg 14760 cagccacccc atctgggaag tgaggagcgt ctccgcccgg cagccacccc gtccgggagg 14820 gaggtggggg gtcagccccc cgcccggcca gccgccccgt ccgggaggga ggtagggggg 14880 tcagcccccc acccggccag ccgccccgtc tgggaggtga ggggcgcctc tgcccggccg 14940 cccctactgg gaagtgagga gcccctctgc ccggccacca ccccgtctgg gaggtgtgcc 15000 caacagctca ttgagaacgg gccaggatga caatggcgac tttgtggaat agaaaggcgg 15060 gaaaggtggg gaaaagattg agaaatcgga tggttgccgt gtctgtgtag aaagaagtag 15120 acatgggaga cttctcattt tgttctgcac taagaaaaat tcttctgcct tgggaaaaaa 15180 aaaaaaaaaa aacttctgag gggtgagttt aaactgtttg ccatgtatca tatgacctcc 15240 cccagggccc agagtaccct aatgtctttg tactcgtcaa tgttttgtgt gggaagggtg 15300 cgggcagtcc ttagggagag ctgagtttcc tctccagact tctgaactgt ggtacgcaac 15360 agcctggcaa aagcagaaac ccagaggcag agatatttag gagaatataa atccccagat 15420 atttgcaaat atgttaaggg agggactctg actttcaccg tccattttta cctcaatctc 15480 aaagggcaag gaggttgaca tctaagttac aaatataatg cacaagcatt tcaaaaagta 15540 ggggaaaaaa aagaaaaaga aaaagagaag cagaaagaag ggggaaaaaa tcactcatcc 15600 ctgccaggca cggtggctca cgcctgtaat tccagcactt tgggaggcta aggtgggagg 15660 ataacttgag tccaggagtt caagaccagc ctgggcaaca tggcaaaacc ctgtctctac 15720 taaaattaca ataattagct gggcgtgtgg cgcatgcctg taatcccagc tacttgggag 15780 gttgaggcag gataatcgct tgaacccggg aggcggaggt tgcagcgggc tgagattgcg 15840 caactgcact ccagcagggg cgacagagtg aaactgtgtc tcaaaaaaaa aaaaaaaaaa 15900 aaaaatcact catccagata gctaggtgtg gcctaaggag cttgcggcct acggaaggtg 15960 tggccgaagg agaggggagg ggtcatcttt aatgatgatg gaggggaggc attggtcata 16020 tacctgagag tcaagctttg ttcatcgtca gatgaaagcc aacttcttcc accagattgc 16080 ctcccagctg ctaggtagtt tcctgtggta catcttaagc ggtaggttgg gataataggt 16140 cccttccagg tcttaaagat gtggattaat aagaacagaa ttttctacaa caataaggag 16200 gatgtcccct attttaggtg aataagcgta ttgcataagc acatgggaag aaaggttaag 16260 aactctaaga ccgtaaattt ccagttgtag aagcctcttg aaggcggaaa gccctttttt 16320 gaaaattata ggcgatatga tctcctccag tggacctagc actgggttaa gagtctctcc 16380 agacttgtca ctgcaagtca cgtacctttt ctaggactaa gtttcttcac tggaagatga 16440 ggaggttgca ctagatgacc tagaaattcc cttccagctt taaattccct cttgtgtcac 16500 ttgctggact tgttctactg gacctggcgc tcccctgctc ctcccttagc tgctacgtcc 16560 gcatcccgca ccagagggcg ccacaggctg gcccggggca gcgtgcggtg ggcggagagg 16620 cagaattagg ggagtctcca ggaggcgtgg tgattggccg ccgccgggcg gaagggggcg 16680 tggggaggga aaggccgagc agcgcggtga cgtctcgctg gcgggggcgg ggccgggccg 16740 cggagcgtgt gacgctgcgg ccgccgcgga cctggggatt aatgggaaaa gttttggcag 16800 gagcgggaga attctgcgga gcctgcggga cggcggcggt ggcgccgtag gcagccggga 16860 caggtcagtc cgagacgaga gaagcggtca ggcaagtggc ggagggaagc gccgggcctg 16920 ggccgaggcg gccagcggga ctggggcgca aggcccggcg gcggggaacg gggtgcgggg 16980 agctgccggg gtccgcggac agcgtcacgg cggcttcctg atgctccggc ccgctccggt 17040 cagccgtcgt gcgttgccat gtaggcgccc cgctgaccct gcgccccccc gcccttggac 17100 ccccatagct gcttgagtga gtccgaaata cgggcctcct tccaccctac tctttgcccc 17160 ctcttgggcc tggtggcttc gtcttctccc acttgtgaga accccgtctc aggcccgggg 17220 gcctctccca ttcccctccc ctgcctctga cctcttcctt cacccgtttc tggtgttcct 17280 gaatccactg ccctatctag cgagagtctt ctctgggatc ttttcctctg tccccctcgt 17340 tatccttgtc tcctgccttc atcctcccat ccttgctctc tcaggcttta ctctctctcc 17400 ccatccctgt ccccttctta tctgtggtct gatccctcct agtattatgg gtccccatcc 17460 tcttccccag ggtgttccta ggctttttcc aactccgtgc ctgcagtccc tgtctcccta 17520 agactctcac cttggttccc tgagaggccc tgtttttgct cctttttccg atttccttcg 17580 tatcaattct gtcccctaca ttcctgtcgc ccacctcctt ttctctccct gttcctttcc 17640 cctgcttcag ggagaagcct tagctccaca ctctactcta ctttcacttg acctgcagtt 17700 gacataacca tggggtgggg gtggaggtgt tcagaccttg atcgaattaa gaggctgatt 17760 gactaaagga aataacagat tgtgtgggag aatgtacgtg ggtgttgcat cagagatgat 17820 gtccaatttg taggccattc ttcttatctc agctttaata cttccccttt atggggctgt 17880 tgacttccag gaacccttgg gggagacggc gctgagctgg atgtgtgcca cgggtagccg 17940 gaggctttgt ccaattcttt tccccttcca ggaaccgggg ttttgtgtct ttccactctg 18000 gatgtgtgga atacacttat accatgatac cccagcctct ctctgccctc actcccaccc 18060 tccaccctgt gctccatccg aaaggactaa gagaattctc cagaagctgt gctccagaaa 18120 atacttgtgg tcctcaacct tttttctgtg tactggctcc agaacttttt tttttcccgt 18180 ttgagttttg aaatagattg ctggcctgat agccctgggg ctggcgggca tctgtcccct 18240 tttattattg gtggcttgaa acagctgctg attttctctc atatctcttt gacaatcttt 18300 ggccaactga atgtgcctct gatgttggtc actgaacatg ttctcttgga gtctcttctc 18360 cctctttttt ctttgttctt ttctgaatct agggaatcca aattgtccct ctttttccct 18420 gggagcttaa tttcctctgt atccaggagg aacggcaatc ccagagctct gttccctttg 18480 cccaggcata gtatgaggta tcagattgat aactaccctg gtgggctgtg gggctgcagt 18540 agttagacct tgtcaggaac ttgagcagac acatctgctt ccagtcacat agatcatctt 18600 ctctggggct tgagagccca ccaccttcca tgtgtgtctg gatgtttaac cttaccaagc 18660 agaagattct cgtcatatcc ttcagtttga gctcctttat ttggcgttgg ttctctaaag 18720 aagtattcaa gaaataacat atcctttgtt tctacccagt ctgatcttgg aggattctga 18780 aagcgtgacc cactctcatg tttagaaatg aaggctcatt gtttcacctg gccccaggtt 18840 tctaggggct cctcttttga tgattgtgct gtctttgagc cagattggac atgggagttg 18900 gcaaccttta ggagctgcgg tggagagcca aaggaaccag cttgaccaga ctttaaagtt 18960 ttgtaatcct cttgtacact gttttctgtg aaagcaatag gattgtggat ggcctgttgt 19020 tgagccagtc ccttctccac tgacccccag accccctttg cagcacttgc aaagatttgg 19080 gtagactgga ccagagggtt ttccttgttc ctgtgaatag catctctcca gggttttagt 19140 ggatcattct aaagaggtag aggtcaagtg ttggacagat ttttagtcta gtctctggct 19200 ttcaaggact cctccttact cttttgagca gtttgttact tctggccgtg cttacccctt 19260 agtagtgctc tctgctgtgg tggcaggtga gagatttgag aacaggtcaa ggccagtcct 19320 tgtggagcag cttgggcacc agcagcccta gatggatagc ttcttgcagt ttgtccactc 19380 tcattttctg agaaggtagt catgattgtc aattaccaaa ttccttcccc attcagtgat 19440 ttttgcagag tctgggtctg gattttcttt ccttctttct tttctgacac aaggttttgt 19500 tctattgccc aggctggagt gcagtggtgc tatcatagtg cactgtaacc tcgaactcct 19560 gggctcaagg gatcttcctg cttcagcctc cagagtagcg gggactacag gtgcacacca 19620 ccacactcag ctaattaaaa aacaattttt ttgtaaagac aggctctcac aatgtctcga 19680 actcctggcc tcaagcagtc ctcctcacct tggcctctca gagcactgag attacactga 19740 ctgctcgttc aggtgccttt ctccttgcct cttcaccatg gccttcttgt gtgatatatg 19800 ccttaaccca ttcaggaaga attgctccta ctcagccttt caacccgacc tgtggtccct 19860 tagacgcctt cccttagggc ctggtaagag gatagacagt gtagagcact agcctgggaa 19920 gccattaggc ctctgctttg ggcctacagc tgtcctgact gttgtgcaag ctgtcttcag 19980 ggcctgcaga ttctggcaca tcctgggata gtgcagctcc tctctctcag gagttggacc 20040 tgtcactttt ttttcgtttt tttttttttt ttttttgaga tggagtctcg ctctgtcgcc 20100 caggctggag tgcagtggtg cgatcttggc tcactgcaag ctccatctcc caggttcacg 20160 ccattcttct gcctcagcct cctgagtagc tgggactaca ggcgcccgcc accacgcctg 20220 gctcattttt ttttgtagtt ttagtagaga cagggtttca ccgtgttagt cgagatggtc 20280 tcgatttcct gacctcgtga tctgcccgcc tcagcctccc aaagtgctgg gattacaggc 20340 gtgagccacc acaaccagcc tggacctgtc acttctaagg gcttcagcta tatcctgtcc 20400 ctgaggccca cctccctgag tgctctctgc aggctgggaa gctgaacagt ggtgccttgc 20460 tccagtgcct ctgaagggct tggaaattgc aggacacagc tttactgctc agcattgccc 20520 acactggaaa cattctcttc ctccctaccc aggtctctgt gggctttgtc cgttctgcca 20580 ggcccttgct tatatgatgt cagaagccct gtgcccggtg ccattgtcaa tgccatgtcc 20640 cagattacct aatggctgct tcttggagat ggtttggtgt ccatgcacat ttttctctgc 20700 cgttggagcc agggtgagga gctgcttgca acagctggat tccagggtgt cagaggagcc 20760 cttggagctt gccttgagcc tggcagggag aacatggccc cttgcatcag gtttcagaca 20820 tatgggtctt cttccttggc agatggctcc tagccttcat aactcagcag gctggtgatt 20880 ggcatgtgac tccagttcat tctgcagggg ctttggtgaa ggcatcaact atgactcagg 20940 acatcttcat ttatgagctt ctacccagct cccttcttac cttgcctgcg ccttctgcgc 21000 cgacctgata aaatcagcaa agccagtgtc tgcttacttg ggagtgagca gactccttcc 21060 aggattgggg aaatatttgc tgactcaggc tcagaatcac atctcagggt ggtgtgtaag 21120 ggaccgacga ggagccagat taccttagga acactctttg agttacggtg ggtataactt 21180 attagagttg ctttgagggc ccctggcacc tgtcagctaa agctgttggg gttttttcta 21240 tcctgcagtg ttgtacagtg ttttgggcat gcacgtgata ctcacacagt ggcttctgct 21300 caccaacaga tgaagacaga tgcaccaacg aggtaatccc attttcttta ctcaggggtc 21360 tctgaccacc actgacacag gatccagatt taagatcctg accttgaaga tgaaataatt 21420 tcactagggc ttattcccag attccaggtt cctgtcacag catctggtac cagttgctta 21480 tgcatagtat aggcatttat taagtaatta ttgaaagtct atggcttaca tagttttttc 21540 aggagcatgg agattcccac ctttgcgccc attaaaagat aaactccaca gggcagggag 21600 ttgttttgct gcctgttgtg tctaggatag tgcctggcac agaggagata atgaataaat 21660 ttttgttgaa tgagtaaatg aacgaacttt cgtctttgcc tatatgggta cctagggtgc 21720 caggccacaa agatggtgtc catcattctc tcttattagg gggcaaatat atatatatat 21780 atatattttg tttgtttgtt tgtttttgag acgaagtctt gctcttgtcc cccagactgg 21840 agtgcaatgg cgtgatcttg gctcactgcc acctccttct cctgggttca agtgcttctc 21900 ctgtctcagc ctcccaagta gctgggatta taggcacctg ccaccacgcc cggctaattt 21960 ttgtattttt agtagagatg gggtttcacc atgttggcca ggctggtctg gaactcctga 22020 cctcaggtga tccgcccgcc tcagcctccc aaagtgctgg gattacaggc gtgagccacc 22080 gcacctggcc tttttttttt ttttttgaga tggagtttcg ctcttgttgt ccaggctgga 22140 gtgcaatgcc gtgatctcgg ctcactgcaa cctccgcctc ttaggttcaa gcgattctcc 22200 tacctcagcc tcctgagtag ctgagattac aggcatgcgc caccacatct ggctaaattt 22260 tgtattttta gtagagacgg ggtttcacca tgttagccag actggtctca aactcctgat 22320 gtcagatgat ctacccgcct cagcctccca gagtgctggg attacagatg tgagccactg 22380 tgcccagccg ggaacaaata tttttaagtg ccaactatgt gccaggcact tgggaaatga 22440 taatagtaaa aaacagatcc aatctctgcc tcatgggtct gtggtttgct ggtatatatg 22500 agagctggcc ataacacaca gaattgtttc ttccatctct gcaggggtag gaggttctca 22560 ccaagagtct gaacaatgct ctgggttccc attgctgttt ccatgttctc aggatgcctg 22620 gcaggtttta taaactctca gaagtggagc tccagggaat acaatgttca ttgtcctatt 22680 tggaaggctg ggttgttgtg ggtccctgct gggggccggg gaaacgtggg cctcctgcct 22740 gatttgtttt aatctctgaa gttcagtggt tccagtagct gtttgtgggc ttcacttccc 22800 cttctctgcc tttaacaccc tgcaggtttt cctgttgtca gacagggtgg tgagtccctg 22860 tgtctctctg tctgtggggg tcaggttgtt tgtagatctt tcaggaaggt cctgggtggg 22920 gggccctcct gctttcaaac ccataccaag tgctttcctc tgaagggaat gtgagggagg 22980 aagaaggggg gagtttcaga gacttctgag gttccccaag agggaagagg tcaaagtacc 23040 tcctgagcgg ggagagctac tgagttgaac tgatttgctt tgccatttgc tttagcagca 23100 gccaggccca gtggcagcaa ttgtacgtgc atttccaggg gtcagttgtc cagttcatcc 23160 ctgagccttg agctcccagt cgcaggtagg aacttctctt ctcctttctt tttttttttt 23220 tttttgagac ggagtctcgc tctgtcaccc aggctggagt gcagtggcgc gatctaggct 23280 cactgcaagc tctgcctcct gggttcacac cattctcctg cctaagcctc ctgagtagct 23340 gggactacag gcgcccgcca ccacgcccgg ctaatttttt gtatttttag tagagacggg 23400 gtttcaccgt gttagccaag atggtctcga tctcctgacc tcgtgatcca ccggcctcgg 23460 cctcccaaag tgctgggatt acaggcgtga gccaccgcgc ccggcctctt ctttctttct 23520 tgattgggta ctgctatatc acctaggacc aggggtgtgt ccaatctttt ggcatccctg 23580 ggccacattg gaagaagaat tattatcttg ggctatacat aaaatacact aacactaaca 23640 atagtgatga gctaaaaata aaaatggcaa aagaatctca tgttttaata aagtttacgt 23700 atttgcgttg ggatgcattc agagctctcc tgggctgcat gcagtcatgg gccatgggtt 23760 ggataagctt ggcctcccta ggcagtcctc tcaccacagc ctcccaagta gctgggacta 23820 caggtgcaca ccactgtgcc tggctaagtt ttttttttgt ttgtttttgt ttttttttaa 23880 tttaatgggt acatagtagt aggtgtatat ttcttttttt cttttctttt ttgttttttt 23940 gagatggaat cttgctcttg tcacccaggc tggagagcaa tggcacaatc ttggctccct 24000 gtaacctcca cctcctgggt tcaagcgatt ctcctgcctc agcctcccaa gtagctcaga 24060 ttacaggcgc ccgccaccac gcccagctaa tttttgtatt tttagtagag atggggtttt 24120 gtcatgttgg ccaggctggt ctcgaactcc tgacctcaag tgatctgcct gcctcagcct 24180 cccaaaatgt tgggattaca ggcatgagct accacacccg gccttgtatg tcttcttttg 24240 agaaatgtct attaaaatct tttgcccatc cttttattag attattcgtt tttttcctat 24300 agagttgttt gagctcgtta tatattcttg ttattaaccc cttgtgaaat gggtagtttg 24360 caaatatttt ctctcattct gtgggttgtc tcttcacttt gttgattgtt tcctttgctg 24420 tgcagaaact gtttaatgtg atgtgatccc atttgaccat ttttgctttg gttgcctgtg 24480 cttgcggggt attgctcaag aaatttttgc ccagaccagt gtcctggaga ttttctcctg 24540 ttttcttgta gtactttcat ggtttgaagt ctcagattta agtctttatt ttgatttggt 24600 ttttgtggat ggcaagaggt aggggtctag tttcattcat ctccatatga atatccagtt 24660 ttccaagcac catttattga agagactgtc ttttcctcag tgtatgttct tggcaccttt 24720 gtcaaaagtg agttcactgt agatgtgtgg atttgtttct gggttctcta ttctgttcca 24780 ttggtctatg tgtctttttt tttttttttt ttttttctct gagacagagt tttgctctgt 24840 tgcccaggat ggagtgcagt ggcgtggcgt gatgttggct cactgcagcc tctgcctccc 24900 gtgttcgagc agttctcctg cctcagtctc ccaagtagct gggattacag gcatgtgtca 24960 ccatgcctgg ctaatttttt gtgtttttag tagagatggg gtttcaccat gttggtcagg 25020 ctggtcttga actcctgacc tcaggtgatc tgcccacctc ggtctcccaa agtgctggga 25080 ttacaggcat gagccaccgc gcctgggcga aagatggctt attttgaagt caactttaga 25140 atattataaa acagcattct caaacttttt ggcctcaaga tccctttaga ttcttttttt 25200 gtttgtttgt ttgtttttgt tttttgagac ggagtctctc tctgtcaccc aggctggagt 25260 gcagtggcac aatctcggct tactgcaagc cccgcctccc gggttcatgc cattctcctg 25320 cctcagcctc ccgagtagct gggactacag gtgcccgcca ccacgcccag ctaatttttt 25380 gtgtttttag tagagacggg gtttcaccat gttagccagg atggtctcaa tctcctgacc 25440 tcgtgattca cccgcgtcgg cctcccaaag tgctgggatt acagatgtga gccactgcgc 25500 cagtcccttt agattcttaa aaattattga agaccccaag ggcttatgtt taccatgtta 25560 gaagttataa taggccgggc gggtgacatt cgttaatatc accactgatc tcatttttaa 25620 aaagtcttta agtattggga agctgtcggg ctcacagtgg tcaatacacg ttttctaaat 25680 ttgagttttc tcttgaaagt ttgattttta tcattggtaa catactgttg gttcttttgt 25740 ttgattttgt ctgagatagc atctccctca gtcacccagg ctggagtgca gtggtgcagt 25800 catagctcac ttgcagcctc caacttctgg gctcaagtaa tcctcctgcc tcagcgtcca 25860 gagtagctag gactacaggt atgtgctacc atgcccagct cattttaaaa ttttttgtaa 25920 agatggtgtc ttgtgatctt gctacatcga ccaggctggt cttgaattcc tggcctcaag 25980 tgatccatct gcctcagcct cccaaattac tgggattata ggcatgagcc accacaccca 26040 gccttttttt tttttttttt tttttttgat atggggtctc actcttgccc aggcttgagt 26100 gcggtggcac agtcttggct cactgtatct agccttgact tccaggctca agtgatcctt 26160 tcatctcagc ctcccaagta gctaagacta catgcatgca ccatcatgcc cacctcattt 26220 tttaacttgt tgtagagatc aggtctccct gtgttgtcca ggctggtctc aaactcctga 26280 gctcgagcca ttcattcctg ccttggcctc ccaaagtgct gggattaaag gcatgagcca 26340 ctgtgcccag ccagttcttt tttgttttgt tttgttttgt tttgagatga agtctcactc 26400 tgtcacccag gctagagtgc agtggtgtga tctcggctca ctgcaacctc cgcctcgtgg 26460 gttcaagtga ttcttctgcc tcagcctccc aagtagctgg gaccacaggc gcgcacccca 26520 atgcccggct aatttttgta tttttttagt agagatgggg tttcaccata ttggtcaggc 26580 cggtctcgaa ctcctgacct catgatctgc ccacctcagc ctcccagagt gctgggatta 26640 caggcgtgag ccaccgcgcc cggccacaaa gaatatttaa aagacagata tcaaaggtgg 26700 agattttaat aaagttagtt tttatttatt catctaggac attcttaaat tggcatttta 26760 ttgtctttca tggttctgtg gattaactgg gcccagctgg gtggttcttg cttgggatat 26820 gtcatgttgc tatagtcaaa tgaagacggg gcaggtgtca tctgaaggct cagctgggct 26880 gggtgtccac gctggcttag gcacatgggt ggcacttgat gctgttgaat ggagtgcgta 26940 tacatgacct ctctatgtgc cttgggcttc tcatagcatg agagctgggt tctgagcaga 27000 agcattccca aaatgaatgt attgagagat tcagaaagaa atgtcaatgt ttctcatgtc 27060 ctagccttgg aagttacaca gtgccacttc ctctattccc ttggtctcag gaccagttca 27120 gattcaagga gaggactaca caagggtgtg aatactaaga ggcgtgattc actgaggaga 27180 gaaacaggcc tccttggaga ccagctacta cttttcagca tccttatgtg aggtaatgtg 27240 gtcttgatac ttccaagaga aaagcacatc ttctcatgat tttcattcct ggaagctact 27300 gttagttagg aaattcctaa taggttgatg gaggacattg aatgctaggt gaaggaattt 27360 gtactctgtt ctttgggcaa tgggggagcc atcagtggtt ttggagcaag gagtggtggt 27420 gttaatattg caaaaaggta gattaggaat ggagatagag aggataggta ggggactgca 27480 gtggtctaaa tgtaaggaaa ttcttcttca cattaaatct gagttcctct ggttgcaatt 27540 taagtgtatt gattttgata tttcagccac acagattaag cacccagagc cagggtggaa 27600 taacccatcc ctaccccata tccatttagg gctgttgatg cacaggagag attaagagtt 27660 cacaaacttt agaaggcaac taaatttagg ttttattgtc aaagcacata aaaagttaag 27720 cataaattat tcaaaaataa aaagcaagaa aggtgaaact tcatttaaca ccactaacca 27780 tgtttcaaat acgcttcagt ttcagtatta tagaagcaag gagtggagcc agaggaagga 27840 atggggagga caaattagag aatggtagga agagggacaa aagtagggga agaaaggagt 27900 caggaggggc acacagcaga aagaatttga aggaggtcag attggagcaa ctacttgaag 27960 ctgagagaag gctccgtatg gggctaacat gttagcaaag agctggtgcc ttctgtgttt 28020 ccctgaggca ctggcttctg ggttttagca tctggtatcc tcaccctttc tttaatacag 28080 acagtcccca acttatgatg gttcaacttt acagtgatgt aaaagccata cacggctggg 28140 cgcatggctc acgcctgtaa tctcagcact ttgggaggcc gaggtgggag gatcacgagg 28200 tcaggagatt gagaccatcc tggctaacac agtgaaaccc tgtctctact aaaaatacaa 28260 aaaattagct gggcgtggtg gtgggcgcct gtagtcccag ctacttggga ggctgaggca 28320 ggagaatggc atgaacccgg gaggcggagc ttgcagtgag ccgagtgcat gccactgcat 28380 tccagcctgg gcgacagagc gagactccgt ctcaaaaaaa aaaaaaaaaa aaaaagcgat 28440 acacattcaa cagaagcccc atcctttagt actcatacaa ccattctttt tttttttttt 28500 ttgagatggc gtctcactct tgcccaggct gcagtgcagt ggtgtgatct cggctcacag 28560 caacctctgc ctcccgggtt aaagtgattc tcctgcctca gcctcctgag tagctgggat 28620 tataggcgac tgccaccagg cctggctaat ttttgaattt ttagtagaga taggatttca 28680 caatgttggc caggctggtt tctaactcct gaccaggtga cctgcccacc tcggcctccc 28740 aaagtgctgg aattacaggc atgagccacc gtgccccggt tctcatacga ccattctgtt 28800 ttttcacttt cagtacagta ttcaattaca tgagatattc aaaattgtaa cataagctct 28860 gtgttagatg atgttgcccg taggctcatg gaaatgttct ggcacatttg cagtaggcta 28920 tgctaagtaa gttatggtgt tcggcaggtt acatatatta catgcgtttt tgacttgtga 28980 cgttttcaac atatgatggg tttgtcctta taagttgagg aacatttgta gccatttcat 29040 cgtctgtgaa gaggcctgag tggattttgt gctggagacc ggtgtgactc agctccagtg 29100 ttgactgacc ttctaaccat gggcttgtga ctgaatgtcc tgagccttca ttttcttccc 29160 tgtgaggatt aaaatgaacc tcaggaaatg ctggcttgtc ctctttctgt caagctgggt 29220 tctgaggggg tgagcccagg taggagtgac ttctgaacca gcccagattt acaggcttgg 29280 gcatagggag cccagcctca gcctgggagc ctcatcttcc attaaatagc tcctggcatt 29340 caggggagtg cgttgcagca ccccattttt ttttcattga agtcctgtct tccagccagg 29400 cacagtgaat cacttgaggc cacgagtttg agaccagcct agccatcatg gggaaacccc 29460 atctatacta aaaatacaaa aattatctgg gcacggtggc gcacacctgt agtcccagca 29520 caccagagtt caagaccagc ctgggcaaca tggcgaaacc ccgtctctac aaaaaataca 29580 aaaattatcc aggcgtggtg gtacatgcct gtagttccta ctccttgggg ggctgaggta 29640 ggagaatcac tcaagccgag gaggttgagg ctgcagtgag ccatatttgt accactgtac 29700 tccagcctgg gtgacagagt aaggccctgt ctgtaaaaaa aaaaaaaaaa aaaaaaaaaa 29760 gtgctttctt cttcttcttc tttttttttt tttttttttt tttttttttt tgagacggag 29820 tctcgctttg ttgcccaggc tggagtgcag tggcgtgatc tcggctcact gcaagctccg 29880 cctcccaggt tcgcgccgtt ctcctgcctc agcctcccga gtagctggga ctacaggcgc 29940 ccgccaccac tcctggctaa ttttttgtat ttttggtaga gacagggttt cacggtgtta 30000 gccaggatgg tctcgatctc ctgaccttgt gatccgcccg tctcagcctc ccaaagtgct 30060 gggattacag tcatgagcca cgatgcccga cctctttctt atataatttt tttttttaat 30120 cgggcagccc tcagaatcac agcagattca gaaagactcc ccagaaaagt tctttcactc 30180 ctaacttcta cccagcctca tggtcacctc agcagctctc ctgagaccgt tactaagttc 30240 tccacagcta atggtaactc agcttggaaa gagttctctt gtaggaggat gctgaaggga 30300 ggttgccaag tggtgtggga aactgcttca tgcttaagtt cacttccata tcatgatact 30360 aaaaggtatc taacagctga ttttcattgc tttcatttgt gatctgctag gaccacttga 30420 tccttatcac tgcttttggc acactcctca tccaagagtt gatcgcagag gcagagttcc 30480 ttgccaccag tccagtggaa gtacaagcca actttgctct ccaaatggca gcagtagctg 30540 agcaggccgc cctgcattct ctcctcataa acatgctgtt ctcaagtttg tcagaatcca 30600 gagctctcta ctcattaccc ccttttccaa acaagcaagc aaagcatgct cttcctttgt 30660 ttcccttaat tgcctaaaac atctccatgc tcatagctgt tgactctctt tgcctcctag 30720 ttcctgttcc tctggtgtag gatttctcag ccctagcact gtaaacatgt tgggctggat 30780 aatcatctct tgtatgggct gtttgtgcat tgtaggatgt tgagcagcat ccctggcttc 30840 tacacactaa gtgccagtga cactcttctc ccctccccag ctgtaaaacc caaatgtctg 30900 cggacattgc caaatgtctc ctagggggca aaatcacccc tggttgagag tcactgctct 30960 atcatttatt cactaaatac tgctggtcag ctgggtccac tggcgaacct cacatcaata 31020 aacctggtaa tagaagcata ttgttcttac attagaagtt tactctcttc agttatctca 31080 ctgggccctt gagggctttg gggaggagtg atgggaaatg gtttacaatt aacagcatat 31140 tccaatgaag aaagaaagca tatgatgtct gcacaacaga ggagagtgtc cacaatgtac 31200 agctcttccc tggagcagtt atgtttctca gggtgtgctt ttccaagtca ttttgtgtct 31260 ggttagaagc caccttccct tagcatccag aggtgccatg tagactaggg ttggtcacag 31320 tgtgtagatt gccattgacc tgcccattcc tagtgtttga cctggtggtc atggcactct 31380 gaccacatca gttttcctct ccaagcccag atgggttctc agaaccctcc accccaagct 31440 gcactcatga gaggcggcac ttctgccatc ctgatctaga gcttgttcat gtggtccaga 31500 ccatttcttt gcagccccct ctcctgtgta aaagccttta ggaaagtgca gagatcctga 31560 tggattcccc ttgaccctca gcacgcacat cctggtatgt agtgctcagc actccgttgg 31620 gggcttcaga ctggagaaga gaaaactgcc cttccctttc aaggacatcc tccaagggcc 31680 agaatgctca gttccgtaaa attaactctc cactgcctac tttttttttt ttttttttaa 31740 gacagtctca ctctgtgccc caggctggag cgcagtggtg tgatcttggc tcactgcaac 31800 ctccgcctcc cgggttcaag caattctcct acctcagcct tccaagtagc tgggattaca 31860 ggcatgcgcc acctcaccca gctaattttt gtatttttag tagagacggg atttcactat 31920 cttggccagg ctggtctcaa actcctgacc tctagtgatc cacttgcctc ggcctcccaa 31980 agtgctagta ttacagtcat gagccaccat gcctggcctg ctgcctattt aaatagcaag 32040 cagttacagt taaagaaaga ccctggcctg gggacgctgc caaggctcct aatctgacac 32100 ttctttcatg tggaccaggg atctgaactg tgtttcttcc aaacttttgg agcttgcttc 32160 cttggtggta agctaaatag tggcccccaa aatatacccc tttataaccc ctgtaacctg 32220 tgaatattat atgacataat ggactttgca catgtaatta aattaaggat cttgagatga 32280 ggggattatc ctagattatc tgggtggacc cctaaatgca atcccaagtg tccttatgag 32340 tgggggccag agagagagat tggacacagg agaaggaggc aatgtgacta ctgcagcaag 32400 atgctacact gctggctttg gaggtggaag agaaggccaa aaatgcaacg aacgtagctt 32460 tggaagctgg aaaaggcaag gaaactgttt tcccttagaa cctctggagg aagtgtggcc 32520 ctgccaacac actgatttta gcccagtgaa actaattttg aatttctgac ctctagaact 32580 gtaagagaat aaatgtgttt tgttttaagt cgctaagttt gtggtaatgt ggtaatttgt 32640 tacagtgcag taggaaacta atacagggct catctgcctc caggtacaag tggtggcttg 32700 gctgatccct gttcttattt taaggcgtcc ctctcatggc acaaggagag cataggccca 32760 gcattttcct gaaccagtat ttgaaacaat ctgcactttg ggggaacctg ttggggagtt 32820 cttggaagca agagagaaag cctcatatgg tacccgatcc ttgctttgga actctcccag 32880 caccttcagt gatatcatga acaaagagtt ctttcagacc agtgaggatg taggattatt 32940 aaaatctgat tctaattcca tcttcccctc tcctcttcca tttattcaat aaatgtttat 33000 tgtgtacctc ctatgtgcta ggcactgggg atgaagggat aaacaaagca gataaaaaat 33060 ctcctgctct cgtggtaact gaggtggggg aagtggaggg agccagagtt aagtcaacag 33120 catgtgagat ggcttcagat gctctctaga aaactaagca gaaaggaggg tagtggcagt 33180 tccacataga gggccgggga aggtctcacc acgaaggtga catttaagtg gaaagaggag 33240 agtgaactat tgtgggaaga atctcgcagg cagagggaaa gcaagttcca aggccctgca 33300 ctgggacaca gctgaggtgt tcaacgcata gcgaggaggc cagcgtggct ggacagaggg 33360 agggaggaga ccaggagatg aagttagaga cgagattggt ggccctgaag tgccataaca 33420 gcttttactt tgcttggcat tttgcccctt ctttctcctc tcctccccct cacaccacct 33480 cacgagtttt aggcccatgt tcctcagttc ctttccatgg gatatttatc ccatcatact 33540 tagggactac cagcaggcaa acatagtcac ttaaaaatat tagaagaggg ggaaaaagca 33600 gcctcctctt tctgaatgat gaaagcagtt ccatggcagt gttggcatgg cactgtgtat 33660 ctagcagtgc aggctggcca ttcaccgcgt agttctgggt ggtttctgag aggttcccct 33720 ccatggcctt cttcagccca tgaggaagtc caacccccct ggcccgctgt ttctttccat 33780 ttcacccctc ttggtctccc cagtgggact cccacagctg acttctatcc ccacctgtgt 33840 ctaatcccct ttccatggct gagccggtag ataacctagt aacgatctga gtagaatatg 33900 gctcccacat gttgaatgca ttgtgctgat cactacatat atcactttga ttcacacaac 33960 aaccctgaga ggtaggaacc attgtacccc ttttagagat gaggatactg aggctcagac 34020 tggttaggaa aattgcctaa agttgcacag ctaatgaatg gcagagctgg gatttgaacc 34080 caagtgatta aagcccatgt ctttgatcac tacatagcag ggccttctag gaaggatggc 34140 tggctggatc tagttgttcc agtttggaaa gtacctgctt gactggccag actgctgttg 34200 gcttcagaaa atagcagtct cttgttggtc actgagaaga ggcaaaattt tggcaggcct 34260 tggggcctcc tgcagagtct cattccaggt cagtggacag aggaggccct gagaatagtc 34320 gtgcatactg gctccctgga tctgctccaa gacagtgagc agcccctaga accacaagag 34380 actggccagc caagctgtgg gcccggcctg tagggtggaa ggaactctta agagaaaaaa 34440 aaaatctctg ctgtgattaa tcaacagctt cagtctggtg aagtgaaaag agaaaagttg 34500 gcccagatgt gcacataggg agagaaataa gggagggaaa ttgcctttat tcatgttctc 34560 ccttaggcca gtcgttaccc cacggatgag atcaccttcc ttttacacca gcctgcaaga 34620 ggtgtgtagt ggtagtctca ttttacagac aaggtgtagg atcagagagg ttaactcgcc 34680 caagtcaaac agctaacaag tggtggaatt ggaatttgag ccctgttctt ctcagttcag 34740 tagccctgct gttcttgctg atgtcacctc agcatgggct ctccaggtat gtgcaaatag 34800 ttatgcaagt gttgcatcca caggcttgtg agcagaggct gaaagggagc cagggctctt 34860 ggtacttgcc ctctcaacat gggccctgcc taagctagca ggactcacta tacccaaacc 34920 agcagtggca ttatattttt ctcttttctt gcaccttgcc atccccgatt tatttttaga 34980 gagccttttc tatttctgtt cccatttctt ggcttggatg caggggctgg ggtgcaggga 35040 tggcgctaga aatcagaaga ggcaggcata gatggcatct cactggtggc cttggctggt 35100 ctctgggaac tgtgcccatc tgcctgccct gctggctcct cctcaggcca gctgtaggcc 35160 ccagggatca tagagctcgg gtagctagag gctgggaggg caggcgagac agagaaacag 35220 ccacacttct catttctttg gcttctcaga gggggatgag cttcctcctg gaacagagaa 35280 gggagtagct cactgcctgt ttctcagggt taggttacgg acttctaggg aatatggggt 35340 agtgcttaag agccagactc tgtgtggcat gggggtgata actgtagacc ctccacctct 35400 ggggattaaa tgagctcata catgtaaagg cacagaggag gcactcactg aatgatgatt 35460 gtttcattag tgtctttaga ccctcagcac ccagcaccat aggtggatgc cagtcattat 35520 tggttagacc aaacagaact tcaaggcagc tgggcatggt ggctcacacc tgtaatacca 35580 gcactttggg aggccaaggt gggagggtca cctgaggtca ggcatttgag atcaacctgg 35640 gcaacgtagt gagacccagt gtctatttat ttaaaaaaat aaaataataa taaaaaaaga 35700 aaaataactt taatgttatt tttgtcgtcc tcttcatctg aacattggcc tgcgtaggta 35760 ttaacctgag tccagtgatt gtttgtggag ccccacaggg tacttcccag tgacagcagc 35820 ctggagtgtg ctcctgccag cccctgcctg ggccctggaa aaggcagggc agtgcagcga 35880 ggaatcttct ggcctaagga tgcctgtaac caccaaacct tatgtttagt tttacacctt 35940 gctgagggtc tggcttttgg atatgagctc tggagtggta ggggtctgtg tcccatgggg 36000 acaggtggtt ctgggtgttg ggcacgaggt gatggaggaa gggcctgcca cttgcaagct 36060 gtttatctga ctagtaataa acagtggatg acacctttag gaaactgtat ttgggagctt 36120 tgaaattaat cagatgccac caatggcatt tttaatttag ttaaaaatgt tttagttata 36180 taaataatgc tcattcgtta tagatgatta gaaaatacag ataagcactg aggagaaaaa 36240 agtaatatat tatcacacat aagtggctgc tttgatatat gatttttttt tcttacctag 36300 gttcttttca aattgtgtat acctagttat tttttctgtc tttcgaaatg ggatgctgtt 36360 taacatttag ttttgtgact tgcctttccc atttttcagg tggttttgtg gtgtctgctt 36420 gtcctccgac ctcctctccc atttgtggga aaggcatttg gcctggaggg aggcacttgg 36480 gaagaactgt ctggttgttc cttgatgtta ttgccagact tgcagagggt agagaggtgc 36540 atgaagtctt actctgcctc tccccatctg tgtctctccc tgtccctgtc cagcgcccca 36600 tctgctctgc ctactcacca ctcctgcctt tttttttttt ccttccatac aaatccagaa 36660 aaggatacca cctccagtct gaaggtttct ttctatggct tttagggatg aatctctcag 36720 gagtttagaa aaaaagatgc acagacagta tataggaagg gtaatgccag ggctgggggt 36780 gggggtcttg ggaagagcaa ctgagaggag ggcctgggtc cctggtgctg tcttgagtca 36840 gtgagaagaa gctgcgtggg tgatgggttg gggcggggta ggcagcaggt gactgcaggg 36900 acctctctga aacagcaagc aagctggcca ggaagacgcc tggagaataa ggggcccaga 36960 taggggccag acatagggga gaagaaaatg tgtctcagtg gcacctgctc ctccaccctg 37020 gccccactct ttccatgatc tttccagaga ttctctcttt ttctgcctga tctgcttccc 37080 tcacctggca gtgcccagcc ccagtagttt gacacatctg gtatatctgg ctactgtgct 37140 ctactgacac ctgtgtggca gcagctgcac agcttcttgg catctccctg ggcagtaggc 37200 agggatccga gcatagagga agatgttcca tgcatcagta actcagattc cgttccagaa 37260 acgcatctca aagcaaggga gaaaattgaa gatggggtat tttgtgggga acaagctatg 37320 ccttcctgtc acttcttaca gaataaaatg gggtctctgg cagtaggcaa ggagaaggcc 37380 tttctgagca ctgagggtca gtggcttctc aggtcttttc tgggccactt tggcctgcac 37440 acaggttggg ctaggaattc atgcttaaaa gcaggctgat ttccataggc tgtgcgttcc 37500 tgtgggcctg cctggatcat atctgttctg ctgtgatccc aggcctggtg agacccactg 37560 gcttcctgta cgtcccctca aagcctgcgc caccgcctct cccttcacca ctcccaagct 37620 ataattaagt tcccatgtga aactgtatcc ctcagctgac acacgcttgt aactacagcc 37680 ctggccattg gacggctcat ctggggtgac ttctggggct ggtgcctcag cctccagacg 37740 ctctccagta ggtctgagta ggtggtgtgg acccaccagg aaagggtgga tccagtgagg 37800 cgggggccag gccttgccca agagtcagaa agtgggaagt cacgggggag atgaagggcg 37860 agtgagcagc tagaaagtgc caggggacac aaaaatgggg gagacaggca gccttttggg 37920 gctggagggc atggacaggg tgactgatgc ctgagccaca ggcagaatgt gagcagttta 37980 ttttatttta tattctacat atttttatag cataaaatta agatatagtt cacataacca 38040 aattcaccac tttaaagtgt gtactttgat ggtttttgta tattaactgt gttttatata 38100 ttatattaaa ttaatatatt tatatattat attaacggtt ttatatatta ttttagctgt 38160 gttgtttata tattatatta actgtgttct attaaccact aattccagga cacttctatc 38220 acctaaaaaa ccctttacct gtcagcagtt actccatccc ttggctgcta atctacttcc 38280 cttctctatg gctttcccta tcctggacat ttcatatgaa tggaattata taatatgtag 38340 ccttttgtgt ctgaacaatt tcacttagca tgttttcgag gttcatccat gttgtagcat 38400 ataatagtac tgtattcctt tttgtggctg agtattccat tgtgtgtata tattccattt 38460 tatttattgg taaattggtg gacatttggg ttgtttccat tttttgactg ctgtgaacat 38520 tcatgtacaa gcttttgtgt ggacatatgc tttaggttct cttgggaata tacttagtga 38580 aattgctggg tcatatagta ttccatgttt aacttttttt tttttttttt tttgagacgg 38640 catcttgctc tgttgcccag gctggagtgc agtggcacga tcttggctca ctgcaacctg 38700 aacctccctg gttcaagcaa tttccctgcc tcagccacct gagtagctgg gattacaggt 38760 gcatgccacc acgcctggct aatttttttt ttgtattttt agtagagacg aggtttcacc 38820 gtgttggcca gactggtctc aaactcctga cctcaggcaa tctccccgcc ttggcctccc 38880 aagtgttact ctttgttttt tgtttgtttt tttttttgag acggggtctc actctgtctg 38940 gagtgcagtg gcgcgatttc gcctcactgc aacctccacc tcctgggttc aagtgattct 39000 tctgcctcag cctcccgagt agctgggact acaggcgcat gccaccatgc ccagctaatt 39060 ttttgtattt ttagtagaga cggggtttca ctgtgttagc caggatggtc tcgatctcct 39120 gaccttgtga tccacccgcc tcggcctccc aaagtggtgg gattacaggc gtgagccacc 39180 gcgcccggcc ccaagtgtta ctcgttaggc agatacctga tacctgtctg ctatactcac 39240 tggtctgcac cttgcttcac ttattatatc ttggccatct ttgcatacca gacatgtaga 39300 gcttccttat tctttttttt tttttttttt ttgagatgga gtctcgctct gtcacccagg 39360 ctggagtgca gtggcgcgat ctcagctcac tgcaagctcc acctcctggt tcacaccatt 39420 ctcctgcctc agcctcccga gtagctggga atacaggtgc ttgccaccac acccagctaa 39480 tttttgtgtg tgtgtgtatt tttagtagag acagggtttc accgtgttag ccaggaaggt 39540 ctcgatctcc tgacctcatg atccgcccgc ctcggcctcc caaagtcctg ggattacagg 39600 tgtgagccac cacgcccagc ccctcattct ttttttttta acagttggct catccattgt 39660 atggatgtgc cagtctcttc tttttttttt ttagacagat ttttgctctg tcacccaggc 39720 tggagtgcag tggcatgatc tcggcttact gcaacctctg cctcctgggt caagcgattc 39780 tcccgcctca gcctcctgag tagctgggat tacaggcatg tgccatcatg cccagctgat 39840 ttttgtattt ttagtagaga cagggtttca ctttgttggc caggctggtc ctgaactcct 39900 gatctcaaat gatttgctca tctcggcctc ccaaagtgct aggattacag gcgtgagtca 39960 ccatgcacgg cctggatgtg ccattctctt attcattgat gaatgtgcag gtgacttcta 40020 gtcttttttg gcaatgaata accttgtcat tttacacaca gataaacaga ttgacaatgc 40080 ttctgtgtga taggtgcagt aatggagata gacactgggg aactagggaa ggcttccaga 40140 ggatagttaa gaaaggctca gggtgagtga gcgctggtat ctgagtgggc cttgcaggcc 40200 tggcagaatt tcagcaggct gagatgttgt aggaagggca aatatgactg aagaaaagaa 40260 caacagctca ggtgtgacag gggaaatgaa gagtattagt tatctattcc tgcataacaa 40320 attaccccaa aacttagtag cttaaaacac tatctcgcag tttctatggg ttaggagaga 40380 tttacacaaa tatgtgaata ccagaaggtg aggattgttg ccagccattt tagaaggctg 40440 actctaatag caagttgcag taaggatagg attggccaag gcaacagatg gagccatgat 40500 tgtggacaat cttacatact tagctgagag gttgtcagag attgatctga gaacagtgtg 40560 agccatggaa ggtctttgag caggagaata atgtgatagt tgctatacag gacaattaat 40620 ctgatagcat ttatgctaga ggggtcaaaa tggagagaga tacacaggca gaggcctaga 40680 ttaggagatg gctatagtag tacaggtgag aaattcctgg cctgtagtac taaatacttt 40740 tttttttttt taagacaatc tcactctgtc acccaggctg gagtgcagtg gcatgatctc 40800 ggctcactgc aacctctgcc tcccgggttc aagcaattct agtgcctcag cctccttagt 40860 ggctgtgact acaggcgtgt accaccatgc ctggctaatt tttctatttt tagtagaaat 40920 ggggtttcac ggtgttggcc aggctggtct tgaactcctg gcctcaagta atccacccac 40980 tttggcctcc caaagtgctg ggattatggg cgtgagccgc tgcatccggc ctctgtatta 41040 aatactttag ctgcatattc tttcttcgaa atgaccacaa aaagaaggta ttactgatta 41100 atatgcatat ggtcttgcgt atggccagac ttatgacttg cgtatggtca taaaggtagt 41160 aagttgcaga gcagcactca gacctgcctg gctttgatga ccgtgccttg atggcactgg 41220 aagtggaaag gtgggggtga ctggggaggt gaggttttgt ccgttccctg gatgctaaaa 41280 ggaaatggag gagtgtaaga ggttgattct ttgaacctgg atggctgggg caaagataac 41340 actgtaacag gaagaagaga ggccggaggg aacactgatg tgatgggaga aggtgagtga 41400 tttgggaaca tggggcattt gttggcaggg tccttcccaa ttatagggcg agtataaata 41460 gttcctgtgc tttgtgtatg aagaggtacc cttggcccaa aaagactcta aatgtggaaa 41520 aaggatatgg tggtgtggtg gtggtttgaa cattgggttt gcgcagatag tgatggtaac 41580 tggcactggc ctgcgcttca gactttcaca gcccgttcac ttctgttgaa actcacacaa 41640 ccctattcag gttatcatca ttccctatct gattgatgag gaaactaagg ctcagaaaaa 41700 tccatttgct caaggagaaa cagtgtttca gttacctact gctgtgtaac aaatcacctg 41760 tgaaactcag tagtttaaaa caaccaccat ttatcctttc tcttgattgt gtgaattggg 41820 tccagatgct tggttctgcc ccacatgatg tcggccaagc ttgcccattt gtctgtgttt 41880 agctgcactg taatgtccaa ggcagcttct ctttgcatgg cctctaatcc ttcaagagtc 41940 tatcctgaac ttcttgcagc atggcatctg ggggccaaga cagtggaagc cagtcgtgtt 42000 gaggcctggg ctcacgtccc agaacatcac ctcaatcatg ttctattggt cagagctgtc 42060 actgggccag cccagattcc aggttgggaa ctagacgcca cctcttgatg gaaagagtgg 42120 caaaaggttt gtagccatat ttaatccaca gacagccggt agtggatacc tggatttgaa 42180 cccagatctg tgtgactcag actcacagtc tgtcatgcca tactgcatcc ctttctgctt 42240 ctggttcctt gggagttgca aagcttgctg gctcttttgt gtggttggag agggctgatt 42300 tttcttgtgg tctcccacag tggaagttgt actagtcatc ccccttttca cagatgagca 42360 tccaagatca ctgtgtctta gggaaccttc caaaataggt ttgctctagg gatttgctag 42420 cttcccccaa gcttcagcag tgatagccag acatgcatgg aatttccctg tggagtttcc 42480 ctttctgcgc ttagtggcct tttgatgcac agtgctcact gggtgggatt cctgctgcat 42540 tgtagccaga tgtggaggct agccctattt tgagttatcc cagatggtat ccagcccagt 42600 gagtcccata cttgactcaa ctggcatatt caccagggag ttttggggaa aatgtagatt 42660 cctgggcctt cctcatgcct attgaattag aattcccagg gatggcgtcc aggaacctgc 42720 ctttaaagct ccccattgat ttgggagtac agcctggttg ggaaaccaat gacttccaga 42780 ggctggcacc ctccctcagc ccagtcattt cagaaagatg cctttgaagt cactgatcag 42840 aggaggccac atgggagaga atgatttgtc cagagggcct cacacttcac aaccaggaag 42900 tgactcctga aagtgtccag gaagtttcta tttgaatctg ctcaaatctt tgatgttgtt 42960 ttgcatgctt tgatagtatc tgagctttat ggtggcctct gggtgttttt ctttgaaagt 43020 gtttgcagag agtagcaagc tcttccagca gtcagcagac actccctaac caggagctac 43080 ttggtctgat ttatttgaaa tatgaaattc ctttgcttgg ctgtctggtt ggtatctttg 43140 gtgtgaagaa tatgacaacc ctagagatac ctaggttagc acagcagttg gacctttcta 43200 gtcttgtgcc ttggacatac ttaaggtaca cttggaaaag tagattttaa aaccctggat 43260 aatttggttt gccagttaaa taaacttcta tattttagaa tatttggaaa ataaagataa 43320 atataaggaa gaccataaaa ttacctagaa ctttcctctc agagaatttc cacggttaat 43380 gttttgaatg gctattcatc cttcccctca tgacccttca caccccctct ttttgaaaac 43440 catattgaag tcatattgaa tgttactttt tagcagtttt gttgagatgt aagtcataaa 43500 gtccacctgt tgacagtgtg caatttgatg gtttttagta tttacagagt tgtacaacca 43560 tcaccacaat ctagttttag aacattttct tagtcccaaa aagacatctt ataatccatt 43620 agcgtgcatt cccgcctcga tgccccatcc cgcccttact ccagcccaag actaccactc 43680 atctgctttc tctacgtaca gatttacctg ttctggacat ttcatataaa tgggatcata 43740 caatatgtga tcttttgtga ttggcttctg tcactggaca taatcatggt actgttttga 43800 aacctgcttt aaacatttat atcctagcaa tttgaatata gttttcaaga atgcactttt 43860 aatagcagag cggtattcat tcttatgaga atatctaatc ttatttaatc agtcctgcca 43920 acagacgttt aggttatttc tcatcttttg ctcttatagc cagtgctctc tgcactcaga 43980 aattattttg ttgatttatt ggttaattgt ccgaatctgt gactagaaca aaagctcttt 44040 gagaacagtc accttctctg tctcccctac tacttggcat agttcctggc acttggtagg 44100 tattcagttt ttaaaggatg tgtatgtgtg tatatgtgca aattttgaat gaatcaaaat 44160 gtaaggatat taataaatgt tgccatattg ccaaattcta gaaagttgta ctaatttacc 44220 ctctcactgc agtataaggg tacctgactc cctgttcctt tgaatctggg ttaatttgta 44280 caggaagtcc tgtgatcttt tgaatgatga tattctctga cctgtcaacc agttagtttg 44340 ggaattgatg ctgtctgtgt cctgaccctg tctgtgggcc aggaagggtc tggctgttgg 44400 gaaagatggt ggtgtgtggg agctctagct gcccgggtcc tcattgtggc tccgtggtat 44460 tgtatcgttt cctgttgccc ttagaaaggc tgcatcgtgt tgtctggcca cttcctctat 44520 tgtgtgcagg catgttgggg agtgagtaga gttgggtgga taaagtatgt gtaagaagat 44580 actggttatg gctgctgatt ggagtgcctt ttgaagtcgt caggagagat gtcactctgg 44640 aagttcagtg tcttcaaaaa tagaaatgaa gaaatgcttc caggaagtct gatgcaactg 44700 gcccagtctg cccccatctg agggtggtgg gcacccacat ggctggtaaa cgcttcatga 44760 gtcacccact ggccacagtc cctgctcctc tccacacact tcctgtctgg aaaagcctgc 44820 tacccgccca tggtgaagag tcaccccaga gaacttgcca gggatgcagg gcaggtcttc 44880 ccaggtcaca gcagcagcct ctcgctcatg ggtttcacag cagttcttct ccccaagatg 44940 agttgtggta actgcttttt agataaatat gtggaatgga tggttgggat tggaaaaaaa 45000 aagagcggct agatggcaag gcagctcttg tgtgtagcag ggagctcaca cgagccccat 45060 ggtggggcag ttaaggcccc agaattcacc ttctccagca gctggctgct cattcttttc 45120 ttcttctcgt ggttttccca tcacatttcc cttggggttt cttccaggcc ttcccctact 45180 caccccacac cctctcagaa gatgctgcct catgtaactt ggccaagctc tcaagcatta 45240 cagatgcctt taccttcttt tctttgcatc ttagaaatca ctcacatgtt ggtctctctc 45300 acataattgc tttttattat tacttcagcg gttgccaagt gcctactcta tgcctagtac 45360 cttgttagac tcgggaatga tgcagaagaa agagactgga ccctgccccc taggagggta 45420 aatgtctgtt ctcccatggc ctctgtttcc cttcctctct gactgcgcct ttgctgccct 45480 ggttcctctt cctctttccg ctgcctgagt gtcaacattc ttcaggcttc tgccctctct 45540 cctctccctc cacgcgcttg tcccttcctg tggctgtgac catctgctct tatagctacc 45600 aaatactaaa gttcttttct cctgaaaacc aggtccctgt gtccacctgc ctgctggagt 45660 gtgcagcctt aaaagcagag tccatctttg tctctgctct ttgcatatcc cctttctgtt 45720 aataatccgt cattctctca gtttcccgga gccttactga cagctttaaa cctcattcct 45780 ctgccatgca caggtcgttc ctcccgttct tttgagtgtt tctccccact gattttcctg 45840 cctgtagtga gttggatggt tgcccctcca aaagatatgt ccaagtccta acccctagaa 45900 cctgtgaaca tgaccacctt gtttggaaaa aaactcattg cagagattct ttcctatgct 45960 gcagacgtac atgaacctgt gctcattgac ctcctctctg ggcctggcct tgttggtctc 46020 tggcttgaaa tagtgactgt acttctagtc cacacagtga ttacatattt attactctgt 46080 ttgagctccc ctagtccaaa ggcaaagttc actgcctgcc ctttccacta actcctaggt 46140 cagacttgct aggttgtgtg cacagcagct acaagtgtgg tttctaaggt gggtctgggt 46200 ttgaatcctg gttctgtcat ttattagcca agggcctttg acagattgta acctttctgg 46260 gcttcagttt cctcttctgg aaaatggagc taatggattg ttgtgagaat tagattgcat 46320 agtgtctagc actgtgcctg acactctgag ctttattttt atttattttt attttttgag 46380 acggagtctc gctctgttgc ctaggctgga gtggagtggc atgatctcgg ttcactgcaa 46440 cctccacctc ccaggttcaa gccattctcc tgcttcagcc tcccgagtag ctgggtttac 46500 aggctcccgc caccatgccc ggcaagtttt tgtatttttt agtagagacg gggtttcacc 46560 atgttggcca ggttagtctt gaactcctga cctcaggtga tctgcccacc ttggcttccc 46620 aaagtgctgg gattacaggt gtgagccacc acgtccagcc cactctgagc tttataacca 46680 gagagatgct gtggctagcc aaatcgctca acctatactt ggaggatccc aagcttggat 46740 gagtgtaaga gctgtgactt aaatccagcc tacttgtatg tcattctgcc acagttgtct 46800 ttgtctctcc acagtttgtc ctggagctgg gcatggtgag ggctaagaca atggattaca 46860 gagctacagg tctaggaggg catgtggaat tatgcctata tgagatagat gacaaagtgg 46920 gtaagagcat gtgctctgga gttagacctg ggttcagatt caggctctat gaccctatct 46980 cagtgacctt gggaaacttt tctttgcatc tctgggtctc agttttctca tctgtaaatt 47040 gggaataaca atggttcata cctgtcttag tctgtgctgc tataacaaaa ttcctgagat 47100 gggtaattta taaacaaaag aaactgattt ctcacagttc tggaggctgg gaagttcaag 47160 atcaaggcat tggcagactt gatgtctggt gaaggctgct gtccacttcc aagatggcac 47220 cttgttgctg tgtcctccag aggggatgaa tgctgtgtcc ttacatggca gaagggtgaa 47280 agtggcccag ctagttccct ggagcccttt tataagagca ctaatccgtt cctgagggca 47340 gagccttcat gacctaaaca cctccaaagg ccacacctct taataccgtt gtattgggga 47400 ttaagtttca acatgaattt tggagggttc acaacattca aaccatagca atgtcttata 47460 gagtaatcgg gatgattaaa tgaaagaatg cccataaaac attgaacaca tgcctgactc 47520 tttgtaaaca ataattatta ggtatcgcta ttatagagac cagcttctgc tgtggagagt 47580 gttaagtgcc ttgatgccac aagccactgg acacgcagct gcatggatca atgtacccct 47640 gtaattatgt aacaagggga catggagaaa aaaacatttc caatgttgcc tcctctgtaa 47700 catgattgta cccctaaact gcacagagga gctttctgtt ctttgaactc catagctgtt 47760 ggtacgtctc ttgtggggtt tgccatagtc tgtcttatac tatgactgtt cttcactcta 47820 cggtacattt ctttctttct ttgtttcttt ctttcttttt tttttttgag acaaagtctc 47880 cttgtgtcat ccaggctgga gtgcagtggt gcaatctcag ctcacagcag ccctgacctc 47940 cccaactcaa gcaatcctca cacctcagct tcccaagatg ctgggactgc tggcatgtgc 48000 caccacacct ggctaatttt ttttgtattt tttgtagaaa tgggatttca ccatgttgct 48060 caggctggtc ttgaacttct gggctcaagc agtccaccca tctcagccta ccaaagtact 48120 gggattgcag tgtgagccac cacgcctggc ctactgtatg tttcttgagg tcaagattgg 48180 gcactgggta actgattact tgaattgaac cttgggattc agccagtgaa agtggtccac 48240 ctggcagtag tgaacctctg gctcctgacc tctgctgctg gtgaatctgt ttctcctatg 48300 gctgcacaga cacagggttt cctgcctcca aagagctttt ccactctgta catgggacct 48360 gaaccggctc cagaaacctt ggagcctggc accacttttc acatcaccca gcaggagcag 48420 aacttagatg gaccaggcat agatgggaaa catgggttta ttgctaaggt catggttagt 48480 tctcggcggt ggggcggggg ggtttagtgg gcaattgctt tttggatttt tgttttgttt 48540 tagaagtaaa aaatatgggc caggcgcggt ggctcatgcc tgtaatccca gcactttggg 48600 aggccgaggc gggcggatca caaggtcagg agattgagac catcctggct aacacggtga 48660 aaccccatct ctactaaaaa atataaaaaa ttagccgggc atggtggcag gcacctgtag 48720 tcccagctac tcgggaggct gaggcaggag aatggcgtga acccgggaca cagagcttgc 48780 agtgagctga gatcgcgccc ctgcactcca gcctgggcga cagagcgaga ctctgtctca 48840 aaaaaaaaaa aaaagaaaag gaaaaatatg tacattttta atagtggtag taagaatttt 48900 ctgtaactac ttacgggaca tcttcactga ggggtctcca gagctcctaa aactcagcac 48960 tcccccagtg attcttacat tttcctgcca gacttgctct tcttcccaca atccccacct 49020 taatggaatc atccacatcc ccctggtcac ctgacagttg ccctctattt ctttattctc 49080 ctcagcctct tttaatcagc caccaagtct tgtttgtcct tcctccctaa tgtcactcct 49140 gttttttgcc gtgaggtctg atggcactag tgctgcctgg ggttgctact gcctcatctc 49200 ttcaaccttt ttgtgtcacg atgtttagga gtttctttca taaacagaat gtggctggat 49260 ttaaatcaaa tctgacaatc tgtcttaagt aatttatgta catttttggg atttaactta 49320 acgtattact gtattatatt ttttagtact ttccatgtat ttccattttt gcctattttt 49380 ctccttccga gttttttttt tttttttttt ttttttggag atggagtctt gctctgtcac 49440 ccaggctgga gtgcagtggt gtaatctcag ctcagcgcaa cctctgcctc ccaggttcaa 49500 gtgattctcc tgcctcagcc tcccaagtag ctgggactac aggcacgtgc caccacacgc 49560 ggctaatttg tatttttagt agagacaggg tttcgccatg ttggccaggc tggtcttgaa 49620 ctcctgacct caggtgatcc acccacctcg gcctcccaaa gtgctgggat tacaggcatg 49680 agccactgct cccggcccgg aattttcttt atttctatac attttacttc cgttgttttt 49740 ttcttttttg agacagggtc tagctctttt gcctaggctg aagtgcagtg gtgtgatcac 49800 agctcactgt agctttgacc tcctgggctc aagcaatcct cccacctcag cctcctaagt 49860 agctgagatt ataggtgcac accaccatgc tcagataatt taaaaaaaaa tgttttctag 49920 agatgggatg ttactgtgtt gcccaggctc gtcttgaact cctcactcaa gccctcctcc 49980 caccttggcc tccaaaagtg ctgggattac aggtgtgagc caccttacct ggcctttatt 50040 tctgttctta ctccaacatt ttctcatctt ttctcccatc cttttactta gaaaaatgtt 50100 aaaattagag gaaatagtac aatgaacatg agtattctct ttaatcttat tcaattaata 50160 tatttctctg tatatgtata tatgcttttg tttggaatca tttgaaatta agtgtggatt 50220 tgttctccat cttcttgctc aggatttggt gtgctgcttt tatctgagga ctcatttata 50280 aaagaaaaaa caaagaaaaa gcctttttac tttaaaatga ttgtagataa acttagcttt 50340 acacagagtt tctgtattcc ttcacccagc ttcccctaat gttaatctct tacatttaat 50400 tctttcttaa gttctggaaa atttcagcca ttgcctcttt ggctttctcg ttttctctac 50460 agtggcctct ggaatgcctc ttagacatat tgttgagcat tttaccacca aagtctctat 50520 atttgttctt tctgtgttcc tttatctgtt taatcatcca agttttcttg tgtagtttct 50580 ttgagactgt cttatttctg attcttggga tgctacttct ctttttgtgt ctgaggcttg 50640 cccctcttgg tggtttattt ccttgtgtgg tttgtaactt tgggtgtgag ttcatctcca 50700 ggagggattc tcttttctgg ctgagacttg ggtcctgaat tatgaaagta tacctcagag 50760 cagcttgttt gcctctggcg gggccatgag tgtttcagtg gtcctggctc agtttttgtt 50820 cactgcttag tgcggcaccc ctttactacc ctagtagtgt aagttcagtt ttatatccat 50880 gggcaggact agcttggtgg cttaagaaag ggtgacctcc cgccgggcat ggtggctcac 50940 acctgtaatc ccagcacttt gggaggccga ggcaggggga tcatgaggtc aggagattga 51000 gaccatcctg gctaacgcgg tgaaaccccg tctctactaa aaatacaaaa aattagccgg 51060 gtgaggtggt gggtgcctgt agtcccagct gctgggaagg ctgaggcagg agaatggcat 51120 gaacccggga ggcagagctt gcagtgagct gagatcacgc cactgcactg cagcccgggc 51180 gacagagtga gactctgtct caaaaaaaaa aaaagaaagg gtgacctccc acccatacct 51240 gacccacaga gccttggaga agtgttcagc ttcttgttgc tttcctttac tgggcagccc 51300 tttgaggctc tcttcttgat actggggctc aaagccacag gcaggcttta aaccgcttca 51360 tttgtgggtg cgaaagtcca caggccattg tggtgtcagt tccactcact gtttatgttt 51420 caattcttct cgcacatagg catttctctt tccaatttgg ctgcatgtct acggacgtca 51480 tttgttttat tttatctggt atttttctgt gatagtggtg gaaaggggaa gctctttcac 51540 gttaatttag tcccgtatcc tactgaaagt ttgttcaaat gcttagcgtg tcatatgaag 51600 cttttcatga tgcagcccat tctacctctc ctgcataaac ttagctctta aattttacac 51660 ttcagcaaat tgaactattt gcataccatg tactgtaatg tttaaaagca caggttttag 51720 ggtccagctg tttggcttgg aacctgtctt ttcataaggt atgattttgg acaagttact 51780 tatcccttcc ataactcagc tttctcatct gtaaaatggc agcgtccggc atgcagtaac 51840 cctgatccct gttggctgcc tctgtggttg ctgcccccag cagttgaggc tgccaggcct 51900 ctgcgcaatt gcactgttta ttcctttgct ggaattgcct gtctcccctc ctctccttga 51960 tccctcccag gagagcagcc tttactcctt cagagcttgg ggtctctgtc tgtagtaact 52020 atcactctgt gggtcgctgc ttgtttacgt atcttttttt ttttgagaca gagtgttgct 52080 ctgtcgccag gctggagtgc agtggtgcga tcttggctca cggcaacctc tgccttctgg 52140 gttcaggcca ttctcttgcc tcagcctccc acgtagctgg gactacaggc gtgcaccacc 52200 atgcccagct aatttttgta tttttagtag agacggggtt tcaccctgtt ggccaggatg 52260 gtctccatct cttgactttg tgatccgccc gcctcggcct cccaaagtgc tgggattaca 52320 ggcatgagct actgcgcctg gccacgtatc tccttttttc agccgactct gacccccttg 52380 agagcagggt cttgtttttc ttggtacttg aaactcctta cattctacct gtcatttggt 52440 gggcattcag tgttggtagt ttaattgcat tgaatttctc tggctgagta gaggcaagtg 52500 ctgggagtgg tatgggacgc agaggcaggg aatgacagca ggaaagcttg ttttttggca 52560 cattttgtgc caagttgatg gaaatgtgaa tcagtatggc tgggacacaa ggtaacaatt 52620 cttagtctag ctgaggatgc cagagactta aaacaagcca gtggccagaa aaagccacta 52680 aatcttgcaa attaaagtgg tttccagact taagaatgca tgccccacat tcccttccca 52740 aactcagcgt ctctgagtgc caggtggagg aatacaaact aattataatc ctagtgggcc 52800 ctgagaatct ttaattttag aataaggctg cacttctgaa aggcgcagtt gctttaggag 52860 taggtgactc tgtatgcccg gggcctccga ggagaccagg atgtgaaaac tcagtacctg 52920 gcatttagag ctgtgtcacc catcacttct gacctcgtta agatttctac cctactgcga 52980 cttaacaact aaaaagggca gatgagatag gtccggggca gagcttaatg gcctgtgtca 53040 ttctcctcag ctttgcctga attctctgga ctcttgagac tctatcatag gaatattctc 53100 agaagagaag gcaatcgaac ctttaaagat ccccagatta tccatcagat aacagtctca 53160 tgagagtgat gaacaatgac agcaccaact ctcaattccc ttggctggaa tttcaggaat 53220 cagcttgaac tcagaggtgg gcacagggtt agggcttgga gtaggcgtct tttctttctt 53280 tttttttttt tttttctttt ttgagatagc gtctcactct tgttgcccag gctggagtgc 53340 aatggctcaa tctctgctca ctgcaaccta cgcctcctgg tttcaagcga ttctcctgcc 53400 tcagcctccc gagtagctgg ggttacaggc atccgccacc acgctcggct aatttttgta 53460 tttttagtag agatggggtt ttaccatgtt gcccaggcta gtctcgaact cctgaccttg 53520 tggtctgccc accttagcct cccaaagtgc tggtattaca ggcatgagcc accgcgcctg 53580 gcttgcagta ggcatctttg cccaacccac gtgaatccca ttagttcttt cttgacagct 53640 ctcagtctgt ggtcatatat taagcaacta tgtgttcagt actgtgctgg gtgctattgc 53700 tgaagatacg gaaatagtaa tgccacaagt ccctcttctt agagaggaga gcttgcactc 53760 tagttggaga gaattcctag cacacagaaa gctgcaacca gtggtgccca acagtaatgc 53820 tgtctgatgc cactgcatgc aagtgtgtcc ctttgacaac ttggtgcttt tgcccaaaag 53880 gagctgttct ctttctgagg cttggactat tcgtttgttt tttctcagat gacttgctgg 53940 cactgttaca gactggtctg aattaactag gactaccttt tatctgtttc tgaggaatag 54000 ccccaaatgt gctctctctt ggtctttatg aacttttcct ttcttccatt tctccaaatt 54060 atgatagatt aactttgcag tgtcatctgc taatctccaa ggtcttctag gggcacgtca 54120 tcagcagttg ctgcttttga ggtatcaagg tttgtcctgt ggagtctcag atagtttctt 54180 tacctgttca atgaatctga ttttccattt ttctaggaga gtgtatctca aagtgattgc 54240 tgaatcagaa tgtctgggag tgtgacttgg aatctattac tgaagattag aggaaaatgt 54300 agcaaattgt gatctgaagc atttatggat ggattgatgg attgatggat agggattctg 54360 actgagttag gtagtctata gtctcagaca gttgtggaag ctccattagg tcatgagcaa 54420 cccagactcc ttccaattct gtgccctgcc attcttaggg tgtggctgct gtcctcatgg 54480 tccaaaatgg ttgccgtggt tccacatctc acagtgagat agaagaagac agaagaatga 54540 tgtctcacct cccttctaag gagacttcag caaagcctgc ctctaccact ttctgtgact 54600 gagggaaagt taattcatac tcagagccaa ttaacctcgt gttttttttt tttgtttgtt 54660 tttttttttt tttttttttt ttttttttga gacaaggtct tactttgtcg cccaggctgt 54720 agtgcagtag cacaatctcg gctcactgca gcctcgacct ttcgggctca agcagggctc 54780 ctccctgctc agcctcccga gtagctggaa ctacaggcac atgcaccaca cccggctaat 54840 ttttaaattt tttcgtagag attgggggtc ttgttatgtt gcccaggttg gtctggaact 54900 cctgggctca agtgatcctc ctgccctggc ctcccaaagt gctgggatta caggagtgag 54960 ccactgcacc caaccagtta cctcatttgt aaaacaggca tggtaacact tagtgcagga 55020 gcaagaggat cagggagcac acatacatac aaagtgcttg gtgctgatgc ccagcctgca 55080 gcacctgttc tctaaatcat agcagcaatt gctgctgcag cacacttctc tttctcctct 55140 ctccaccgct ccatctctcc agggtctcta ccttgcctgt caggctccac tttcctttgc 55200 tcctgccttt tgcatctgtt cctctctgca ttctcccctc atctcctctc ttccgtgcac 55260 ctcattttcc tgctcccctc ctgcctctgt ccctcatctt cttttcagag acttaaaacc 55320 agccagtggc cactggcttt tcagtgtctc ctcctcctcc ttgaactacc tcttccttct 55380 agtgcttctg tccttccctc ctcctgacct ccagtgaaca ataattattt gtaacgtaat 55440 gacaaattat cgttacgtta caaagaccag aggttagagg tggtgattgg ccttgtaaac 55500 tgtaggtcag cgatgagtaa tttaagaaaa ctaaatttaa ttggtttact catgagtcag 55560 ttaggaattc tatttggcag ctagaacaga aatagcagtg gcttaaacca gtagaaattt 55620 tttctattgt gaaaagaaat ctagggggac agtctagggc ttgtctctcc gctctgccat 55680 ccttagtgca gggcgtccaa ccccaaggtt gtatcatggt ctaagatggc tgcgggagcc 55740 ccaaccatca tgtcaggttc taggaagaaa gcaggaagaa gagaggaaag gcaaaaagaa 55800 cacatgctcc atcttaagga gctttcctgc aagtcccacc aaatagcttc agcttctcct 55860 ccttgcctcc attagttgca agggaggctg agacatcatt tagctgagca cattgctacc 55920 tgaggtagaa ttgggattct gtcaccaaga aagaagggga gaatggatat tgtgtaaaca 55980 attagcaagg tctgccgcca ttcattcaac attaatttag cacctacaat ctactagtta 56040 tgaagaacag tcaaggtgcc ccattccaga gggaagacat agataagtaa acaagtacag 56100 tacacaggat gggtgaggaa gagggggaga gtggagaaag gcatgaagtg ggtatcttac 56160 ataccctcca agggcgcatt ctagagttcg tcatgcagac attcttgcct gtggtttgtt 56220 tcctcttctg ctctctcctt tttgtcattg tcgccattgt tgttgttggg gtttgttttt 56280 gcccctccat tctgacgtca gcagtagccc ctagaacctt aggaaacaga aacagttggc 56340 ctagtgggaa gctctgggat gggagccatg tgtttgaaga gccaagtccc agggccatta 56400 ggcaattaga ggcctgtggc aggagagcat tcgttcctgg acatgccgtg gcactgccag 56460 gagactttac tccaggaatg gggtcgttgt caggaggcat gttctaacca ggcacctggc 56520 atcacctata ctgtgcttgc cataaaatga aattttggag cagtaaaatt ctccaggtcc 56580 cgtgaaatgg gtataattgt ttcacccaca ttaatttaac cagaggtctt ggtatgctat 56640 agtgttaatt atgtgtaaaa atacttctac ttactataaa atgtattgac agttagacat 56700 tgatattcct tcttagaact caaaaattct aggaataact tgggagaccc acattatctt 56760 tcattttgat cccaagaaca tctattgctg tttcctttgg gaagttagag aggcaatggg 56820 aagcctgctg cagagatttt ctaaacaggc atatcccagc ccctaaggag tttgaaagtg 56880 tcagagaagg ctaaaggatt aaaagatcgt ttgttctggc acagtgcaat ccttatgagg 56940 ttgtactgtg accctgggca agttaccatc ctatcctcag tttcttcatc tgtcaaattt 57000 tgatcctgtg gaccctcccc aacagggctg tgatggaaat gaagagacac atgggaagca 57060 ttcagtatgt gttgttgtta ttatcatcat tattgttatt tttattatta ttagtgctac 57120 tggtagtgct agcccactag cccactgggc agcgactgca cccgtatcag agtgtggaaa 57180 gccttggatg ccacattaag gagttggatt ttgtgggcaa gggtgtgaca ggtcatggct 57240 gggcttagaa atgtcactga caggagtgga gaggcagatc taggtgagga gtgggcaggg 57300 cttgaacctg gccagtcaag gaaaggggag aatggatacc tgagtggtga gggaggaccc 57360 actggtgctg gagttcagcc tgtgtgccca agggatggtg tcccattgac ttgggtggga 57420 agcactagag gtggagcagt tggggtggca ggaggggaaa gaagcatatg gtgatcagct 57480 cagcaaacaa atgggtaact tgcgcactca gtgctcggtg gtgcccaggc tggagtgcag 57540 tggcgtggtc ccggctcgct acaacctcca cctcccagcc gcctgccttg gcctcccaaa 57600 gtgctgagat tgcagcctct gcccggccgc caccccgtct gggaagtgag gagcgtctct 57660 gcttggccac ccatcgtctg ggatgtgggg agcccctctg cctggctgcc cagtctggga 57720 ggtgaggagc gtctccgacc ggccgccatc ccatctagga ggtgaggagc gcctctttcc 57780 ggccgccatc acatctagga aggaggagcg tctctgcccg gccgcccatc gtctgagatg 57840 tggggagcgc ctctgccccg ccgccccgtc tgggatgtga ggagcacctc tgcccggcca 57900 cgaccccgtc tgggaggtga ggagcatctc tgccccgccg ccccgtctga gaagtgagga 57960 gaccctctgc ccggcaacca ccccgtctga gaagtgagga gaccctccac ccggcagctg 58020 ccccgtctga gaaatgagga gcctctccgc ccggcagcca ccccgtctgg gaagtgagga 58080 gcgtctccgc ccggcagcca ccccgtccgg gagggaggtg gggggggtca gcccccccgc 58140 caggccagca gccccatccg ggagggaggt gggggggtca gccccacgcc ccgccagccg 58200 ccccgtcagg gagggaggtg ggggggtcag ccccccgcca ggccagccgc cccgtccggg 58260 agggaggtcg gggcgtcagc ctcccgcccg gccagccgcc ccgtccggga ggtgaggggc 58320 gcctctgcct ggccacccct actgggaagt gaggagcccc tctgcccggc cagcggcccc 58380 gtccgggagg gaggtggggg ggtcagcccc ccgcccggcc agccgccccg tcagggaggg 58440 aggtaggttc agccccccgc caggccagcc gccccgtccg ggagggaggt cggggcgtca 58500 gcctcccgcc cggccagccg ccccgtctgg gaggtgaggg gcgcctctgc ctggctgccc 58560 ctactgggaa gtgaggagcc cctctgccag gccagccgcc ccgtccggga gggaggtggc 58620 ggggtcagcc ccccgcccgg ccagccgccc cgtccgggag ggaggtgggg gggtcagccc 58680 cccgcccggc cagccgcccc gtccgggagg tgaggggcgc ctctgcccgg ccgcccctac 58740 tgggaagtga ggagcccctc tgcccggcca gccgccccat ccgggaggga ggtggggggg 58800 tcaggccccg cccggccagc cgccccgtcc gggagggagg tgggggggtc agccccccgc 58860 ctggccagcc gccccgtctg ggaggtgagg ggcgcctctg cccggccgcc cctactggga 58920 agtgaggagc ccctctgccc ggccagccgc cccatccggg agggaggtgg gggggtcagg 58980 ccccgcccgg ccagccgccc cgtccgggag ggaggtgggg gggtcagccc cccgcctggc 59040 cagccgcccc gtctgggagg tgaggggcgc ctctgcctgg ccgcccctac tgggaagtga 59100 ggagcccctc tgccaggcca gccgccccat ccgggaggga ggtggggggg tcagcccccc 59160 gccgggccag ccgccccgtc cgggagggag gtgggagggg tcagcccccc acccggccag 59220 ccgccccgtc cgggaggtga cgggcgcctc tgcccggccg cccctactgg gaagtgagga 59280 gcccctctgc ccggccacca ccccgtctgg gaggtgtgcc caacagctca ttgagaacgg 59340 gccaggatga caatggcggc tttgtggaat agaaaggcgg gaaaggtggg gaaaagattg 59400 agaaatcgga tggttgccgt gtctgtgtag aaagaagtag acatgggaga cttttcattt 59460 tgttctgtac taagaaaact tctgccttgg gatcctgttg atctgtgacc ttacccccaa 59520 ccctgtgctc tctgaaacat gtgctgtgtc cactcagggt taaacggatt aagggcggtg 59580 caagatgtgc tttgttaaac agatgcttga aggcagcatg ctcgttaaga gtcatcacca 59640 ctccctaatc tcaagtaccc agggacacaa acactgcgga aggccgcagg gtcctctgcc 59700 taggaaaacc agagaccttt gttcacttgt ttatctgctg accttccctc cactattgtc 59760 ctatgaccct gccaaatccc cctctgtgag aaacacccaa gaattatcaa taaaaaataa 59820 ataaatttaa aaaaaaaaaa aaaaaaaaac aaatgggtaa cttgctgcct gccagggact 59880 cctgtggcct ggcatgaaga taattgactg tgttttgaac gcattataag gcccctgcga 59940 ggcctccgtg tggtatttag acagtcagca ggagagaggc cccggacagt ggtgtgggat 60000 gcagtcccgg tgtgtgaaat tgccagtggt tggattgtgc agcggatgga ttgagcaggg 60060 aagccagcca agaacagaat ggggtgggga gggcgttggg ggtaggaaag gaggatcatc 60120 atttcaaggg cgggtgaggg aagaacaagc agcagagtgc aaagccccca gagggaaggg 60180 cgatccttat gaggttggac tgtgaccctg ggcaagttac cctcctgtcc tcgggaggaa 60240 gtggttgaca gaatcattgc agcagtgaga tggaagcagg accagggcag cagccaaggg 60300 cctcttagat ggcggggcga gaggaggtca tccaggacct ccaagagaac tgtttcaaaa 60360 tgggaggaga aagcagtgtt cctctggtgg tggttctaat gctttgtatt tgtagttctt 60420 agtcgttttc aaggcccttt gcattctttg catcgtgctc tcttaccacg atgcatagtg 60480 agaccccacc tctacaattt tttttttttt tttttttttt cagtgagcca ggtgtggcag 60540 tacacacctg tagtcccagc tacttgggag gctgaggtgg aaggattgct tgagcccagg 60600 aggttgaggc tgcagtgagc tgtggtcatg ccactgcatt ccaacctggg tgacagagcg 60660 agaccccatc ttaaaaacaa acaaacaaac aaatacttgg ttcatttggt acttggcttt 60720 ttggatcttg gggaatattg gagatgacgg aataacttca aggagactaa cttcaaggtc 60780 aagactcctg ccaagactcc accagacata aaataggaaa atgatgatag aatctacctc 60840 actgttggcc tggcagtggg agtttgttga taaccctgaa gatgaactcc tcaaggtgaa 60900 gagcaaggct tcagaatggg ctgctctata gatgtgagag acagagacag acctagcctt 60960 gcaaaaaggt tgctgtagtc tttctcctac catttccagc acaggatggt ttctggtccc 61020 aggaggccct gtcttgtgct gtgtgggacc cttagtgctg tgaagactag cagcaccctg 61080 gaggtctttg ttccttctgc cctttccctt agattgcatc tcctaacctt ggtttatata 61140 tttgtcagga ggtgggggaa acctgctttc ccccacctcc tgacaaatat ataaactgga 61200 aattgatcca gtcctggatc agtttgggtc acacaacagg gtcggcagtg ttggcccaca 61260 gagggccctg ggccccttat ttcccctttg gccctaggct ctgagctctc aggttttggc 61320 taccaagcaa aaggtggagt tgcatcagtt gttgctcttt accaggcccc caggggttgc 61380 tgtcggtgag ctcacaggat gttttcttct ctctgctttg acttgtgctg aatttctgac 61440 tgaccccatg cagcatctgc gtggcttctg gggcccgagc ccaggtttca gcaagctgcc 61500 catggtttac cctcaggatg aactctagga agctggggag cactgtggtg gtggtataag 61560 ttcagcccct cattttagtt tatttttatg ttaaaatgtt tattaataca aaataacaca 61620 tagatcagaa tgttgtatga cagaaaggga ctgactggac tcaggatctc ccaccccagc 61680 cccaggggtg aaggtgaagg tgattggttt cctgggtatc gttccttcca cggtttttcc 61740 atgcacgctc tcatgtgcgt gtgtttttat cctcatcctg ctcttcaccg tggcagtggg 61800 gagcacacta aattgctctg acacctagtt agtttttttt ttgattgagt gtatcttggg 61860 aatctttcca tatcagcata tacagataca ctgcattctt tttttttttt tttttttttt 61920 tgagatggag tcttgtgctg tctcccaggc tagagtggaa tggcacgatc tctgctcact 61980 gcaacctcca cctctcaggc tcaagcgatt ctcctgcctc agcctcccaa gtagctggga 62040 ttacaggcac ccaccaccac gcctggctca tttttgtatt ttgggtagag atggggtttc 62100 accatgttgg ccaggctggt ttcaaactcc tgacctcaaa tgatctgccc acctcggcct 62160 cccaaagtgc tgagattaca ggtgcgagcc accacacctg gccattcttt tttttttttt 62220 tttaaagctt ttttgttgta catatgatgt atgttaggtg gactgtggct tataaaacct 62280 cttcatagct gttttccagt gcgatccttc tagccctgtg aggaagacag accagaagaa 62340 attttttgca gcttaggata ctctgcaaac aaggaggaag tgagaggagt tggggctcat 62400 ttaatgcaga ctaaaacagg atctttcctt tgagagactg agacaggatg aggagcaggc 62460 tgtagatgtg cacacatatc cactcagaga gaacatgaga ggcagcatct gcagaggagc 62520 ctggtgggca gcacgtgccg cgggggcagg aaggggagag cttccgggaa ggggatggaa 62580 cttgagccaa acctttttct caaaaagagg ggaggccggg tgtagtggct cacacctgta 62640 cccagcactt tgggaggctg aggtgtgcag atcacttgag gtcaggagtt tgagaccagc 62700 ctgggcaaca tagcaaaacc ccatgtctat taaaattaca aaaagtagcc gggcgtggtg 62760 gcgcacacgt gtaatcccaa ctactcagga ggctgaggtg ggacgatcac ttgaacctgg 62820 gatgcggagg ttgcagtgag ccgagatcgc gccactgcac tccagcctgg gtgacagagt 62880 gagactctgt ctcaaacaaa agaggggaag gtaggaatga gacaaaggaa aaggggaggc 62940 gggcatttca ggcaggagag cagcataggc agaaggtgca aacatgtggc agagggtcgg 63000 tgtccatgga cacgcttcca ccacctggtc taatcattga cttatcagac atctaaatgc 63060 ctgctgcgtg cccagcactg tagagggcca caaggcagca ggaacctctc agtgcctgcc 63120 ctctgtgggc tctgactcca accaggagaa actggacgca caacagcagg caaaactagc 63180 tgatggtgat agaggtcaga agagtggttt acagccaggc acagtgactc acacctgtaa 63240 tcccagtact ttgggaggct gaggtgggag gatagcttga gcctaggagg ctgaggctgc 63300 agtgagctgt gatcaggcca ccactgcatt ccagcttggc ctggacaaca gagcaagacc 63360 ctgtctcaaa aaaagcggag tagttactta ttgtggagat tagctgttca ggggcacaaa 63420 gaaccttgta gggtgctgga aatgtttact gtcttgacct gggtgatggg tttttttggt 63480 ttgttttttg tttgtttatt ttgttttttt gagatggagt cttgctcttg tgccccaggc 63540 tggagtgcaa tagcactatc tcggctcact gcaacctcca cctcctggct tcaggcaatt 63600 ctcctgcctc agcctctcga gtagctggga ctacaggcgc ctgccaccat gcccggctaa 63660 tttttgtatt tttagtagag acaggggttt caccatgttg gccaggctga tcttgaactc 63720 ctgaccccag gtgatccgcc cgcctcggcc ttccaaagtg ctaggattac aggcgtgagc 63780 caccgcgccc agcctgggtg atggttttat ggtggtatat aaatgtagga attcatgggg 63840 cttattatgt ttaagattaa tgcagattgc atactctaac catgagtatg ttatacccca 63900 attataaatc aagcaggcaa ataaatagca tattccccaa aataaagaag taaaatgcaa 63960 tagcaagaaa gtgtgcaaag agctatgcct gggaaaatac aggaactgta tcattttgaa 64020 agggtgagag atgagatttc agaccctcac cctctctcac tgtgtcattt ctgaagggcc 64080 ctggtgactt tctttgcccc tctccactct tacagtcatt ggaaccagag ggcagaaccc 64140 cgggcctcaa aattaagtct atacacctta gaaactagga attcaaggct agccaggtcc 64200 tggcccttta tctgtccttc agagagtggg gtgccccagc caaagcggac agctcgccat 64260 tcccagatgt gccctgtgtt ctcggctgcg ttgcctgact caccttggcc ttccctggga 64320 gtgccttttc tagttttctg gcccagctag aagaaagggc cattacacct actccagaat 64380 catccctgct gctcccaagt tggaatattt cttcctccac agtggccacc tcttatgtgc 64440 cctccatcac acagtttgta tacttacctc ttcaagggca ggctgtgttt tactcctttt 64500 gtcatctctc cccatagggg tctaggtcag tgcttttgta catggtgcct gaaacagtcc 64560 cataaatgat attttatttg agtggggctc tctggctgga gctactaggt ggaatgttgc 64620 catgtgggag caggggtcat aacgaaggct ggccatcaag atgctggtac ttccaaattc 64680 ccaacagtta ttaactttca ggattgagga acaagtaata gaaattccct ggaatgccca 64740 aaatcataac tttttctata tctttcctaa cactttatga gtttttaaat aaggagtggt 64800 cataataata aatggaatta ttttttctgt gataaaattt cagaggtgtt tctgtaaaaa 64860 aaaaagaaag aaaagaaaaa gagaaaaaaa gatgtaacat taaatgcgtg gtttggtggt 64920 ttggttgggc acagtggctc acacctgtaa tcccagcact ttgagaggcc aaggcaggag 64980 gatggcttga gctcaggagt ttgagaccag cctgttaaca gagaaagatt ttgtctctat 65040 taaaaataaa aaaaattagc cagcatggtg gcatgtgcct gtagtcccag ttactcagga 65100 ggctgaggca ggaggatcac tggagcccag gagtttgtgg ctgcagtgag ctatgatagc 65160 accactccgg gtggtgacag tcttggtgac agagtgagac cccatctaaa aaattaaaat 65220 aataaaaata aaataaatgc atagtttatt gttattatgg acaaatatat tgtgatatat 65280 gattgtatat atttcattag catgtaaaaa tgatctctat ctgatataga catctgataa 65340 tagaaaatgc ggaagagagc attaacacaa aatgaatacc aaataaaaat cacttaatga 65400 tttaagagta atactacaca tatttaatat ttaaaagtta ctcttaaagg ttaaaactag 65460 agaaaagtta aaagaataaa acaatgaatt tctgtatttc cttcaccaag attcaccagt 65520 tgctaacagt ttggcgcatt tgcttgatgt gtcatagatt gtccacattc catatctgtt 65580 ggattgtttc cttgtgatta gattcgggtt tacgttgttg gtgggaaccc acatagatga 65640 ggtgtgcttc ctgttgctta catcaggagg cacagtggtg ccagttggcc ctagtaaaag 65700 tgatgctaac ttgattcacc tagttaagtt ggcatctgtg aaggctactt ttgtattaag 65760 gcagtgaact tcgaaaggaa cttttccctg actttgggaa ttgctgacag ccatttagga 65820 aaagagacga ctgttttctc tgtcacgtgt agctgcagtt aagaaacatc ctagtatagt 65880 cagacaagct ctggaagcca gtatgtcagg acctgggacc aagctctaca gccatctctc 65940 cctgtgacca tagccaagtc atttctgagc tatgtgagac tgtacgtata aggaaataga 66000 cactcagaga agtgaagcag tttgcccaaa gctactcagc tagtgagtga cagagctagg 66060 attcaaaccc atttttgtct ggggacaaag ccctgccctt gtccttatgc caagctgcct 66120 cctttgtgag gcagctggtt gttatgattg tggcactgac cactcagatg ttgtgctcgg 66180 aaccaagcag aagagcacca agccttatag gccctgacag gtgagaggga agagagaggt 66240 gccttccttc atccatctgg actgcccttg gaattcctgg gccaatttgg ggaaccctgg 66300 caccaaccct aacatgggcc agtgccagca ccaggtatat gcagcctgta cttcccagtc 66360 tgggcaggca aaggctggaa ccgcaccagc agctctcctc tgtaacgcag cacggaagta 66420 tttatttcaa gggtggctca gagaagtgaa gcagtttgcc caaagcaaac tggttcctcc 66480 cctgcatgtg gcaggctgtc ctgcaagctg ggaggcgggc tgttggacgc tccccagcca 66540 cccttgaaat aaatgcttcc atgctgtgtt gcagaggtga gttgctggca ccgttccagc 66600 ctccgcctgc ccagactggg aagtacaggc tgcacatacc tggcactggc ccactttagg 66660 gttgatgcat gggatcccca gattggccca ggaattccaa ggccagtcca gatggatgaa 66720 ggaaggtatt gcctcagcct gcctgggttc ctcgggggag agagggagac aggaagcatt 66780 aatgggggaa aggtgtgcca agccttgcca agcaggtcat tgtgggtggg gagacaccgc 66840 ctacgtgctc gggacgcccc aggatttggg tggaggttct tgaggaggca gcgagaaagg 66900 agagctgtga cagcgactct ggagcactct agaatggctc ttagtgacct tctgctctgg 66960 tgacccacca gcagcccacc aacctgtcat cttccagtta gcagccatcc ccagggccag 67020 tagcacctga gcacttggta agtagaaaga cccagtgtct gctcctcatg ttaccctggt 67080 gtccttcccc agagcagttg ttttgggaaa aactggctcc ggctgaagca ggaaatggtg 67140 ctccttgtca aggtcatgtt gtcctgttgc tatagaactg agagaccctg aggatcagcc 67200 ctctgaggtt cctgttacct ctctttacac tattcatcct cataggcctc caggaagggg 67260 gccaaattca gggccaggac cagggccagt ggcttggtga ggcataggct ttgctgtatg 67320 ggccctgtgg ggagctctgg ggagttctta ttcccctggc tcatggccac catcatggag 67380 agacccccag ggctctggca gccaagtggg caggagcgga gggagggggc atcttgggag 67440 ggagagcccc gaaaggaaac agggtttata gacagactcc cggtcatcac tgcttgtaat 67500 gcttgctcag tgaacagtgg cagtgctttt cctgactctt tcaagagagt cccttctctc 67560 tcctgctagg tgggctgaga tccagactgg ttagaagcct aacatgacct ggcgtgtgag 67620 accttgcact ctctgaatgt attccccaag tgtcatcaat aagtcactcc ggttctggca 67680 tttccctatc ttatcctgca tacccctcct cctggctctg agtcctgagg agtgagattg 67740 cagtttttga cccatgttct tactgagaag tgggctgtca tgcaggtata atataaaatc 67800 cagaagctgc tgtcagaatt atgtgagaat tgggaggtga cagcatgggc caattttctg 67860 aatctctagg gtgctttgat gcttaagaag tctttctggg ctccctgaga tttgaaacct 67920 cttgttttat ctgtcctttc actctggttc ttcctttcct ttcttgggga cttcatgctt 67980 ctcaaatatt tgttaagtgg gtgcttatat gggaccttag aaagcaacca gctaggcatg 68040 cacttggaaa gggacaggag gactgagaac aagactaaac atttctgtga gattgagcag 68100 caggagagct ttgcttcttt actggtgtca gtatgtgcct gcagctcctg ggctgccttt 68160 actcgagtga gatccattta caagactcct gactcttcta ctccacttta ttacaaaaga 68220 tgttgatgct cgttttaaaa acaatttaaa ctacaaacat gaataaaggt atttatctcc 68280 ccaccagtcc ccctcattct cccttccctc ctgttcttag actggtttcc cctcccagta 68340 gtttaggttg tggggcaagt gtgtgtagct ctgcatcagt gtcttaaaca aaatgagtgt 68400 gtcgcggatg cgcagctcgg ccacttcctg ttttcttcgg ctgtatgtca cggccatcct 68460 gccatggcac catgtctgtg ccacctgctg ctcttgggca tctgcagaga attccatgac 68520 atggatcttg aggtagtctt cttcagcttt ttctacttca tcttattttc tacaatcatt 68580 tatatctgct tgtaaaacct ttttttaaaa aacttgctta ctagacacag cacatgttca 68640 ttttagaaaa tgtagaaaaa tcagataagc agccgggcat gatgactcat gcctgtaatc 68700 tcagcacttt gggaggccaa ggcaggcaga tcacctgagg tcaggagttc gagaccagcc 68760 tggccaacat ggtgaaaccc catctctact aaaaatacaa aaattatctg ggtgtggtgg 68820 tgcgtacctg tagtctcagt tacctgggag gctgaggtgg gagaatcact tgaacccagg 68880 aggtggaggt tccagtgagc cgagatcatg ccactgtact ccaacctacg tgacagagtg 68940 agaccctgtc tccccgcccc ccccaccaaa aaaaaaaaaa atcagataag caataaaaaa 69000 agtagaaatg acccctaaga ggtaacattt tggtgtgtat aatacaaatg tcttttacat 69060 ttgctccctc atctcagcat tgccactgcc attaaagaag gcacatggtc attagtccca 69120 tctcacagat agaaagcctg agccacagag agggtagcag gttgcccaga ggtacacagc 69180 atagtaaaga cagagctggg ggtgtacctg tcatataact cagcactaat gctctctcca 69240 ctgtagccca cttgctgtct tcgcaccttg ggacagagag gggggtgcca ggcattggat 69300 ttcagatgca ttgaactgcc ttatccgaag gttaatagac agatgactgc ttgaggaaag 69360 cccctgattg acttaggaga agaggtagaa gacacgctct tcaggtttac agacaatatg 69420 agacaaggga ttgtttgcgc tttagagaac agaatcgggc ttagaaaacc ctcaaagagc 69480 tctaatgaca ggtcaggagc aacaggataa actttggaag gggtggatgt aaaggggccc 69540 ccttctattt ccgtaagcac aggaaggcct gacttgatgt tcaggcagtg atgactggtc 69600 tgcctgggag tcatagtgga ctgtaagcag ggggacatgt catttcaaca ctttaacttc 69660 atcttacttc ctgggagtgt taggagccac acatcaagag gacgtgatga actaaggaag 69720 agggtttggg aacctgtcaa atgacaaatg ctgcagtgac agaggtttca cctagagaag 69780 atgggtttgg ggaggaaaag gtgcaggcca agacttggtg accagggcca ggcaggacag 69840 gaccaggaat ggcagttagt tgaagggaga gtctaattgg tcaggataca gaacaccttt 69900 ttagcattag agttcaggaa tagtagcgag aggcagtaca gtagagtggt taagagctcc 69960 tgctctgatg gcttactggc tcccggctcc cagagtcttg gaagttacct cactcttcca 70020 tgttccaatt ttctcatctg taaatgggga taatgggtac ctgccttata gtccacttgt 70080 aaaacctaag ggagttactc catgaaagca gcttagaata atgcctggca cacagaggtg 70140 ctgtgcactg tttgattcac acggaagctg ggtaggctcc atggaagaag agttgtagaa 70200 ggtcttctgc atgcatttgg tatgacctgc ttacccagcc ttttccattc cctgcctccc 70260 tgtctccttc tttctttctc catcttcctc tcctgggtct aggatcctac atttccccta 70320 cctaggacac cctacccctc aatctttatt ttcttctttg aaactcttaa gtattttatt 70380 tcttcttctt cttctttttt tttttagaca gagtctcgct ctgtcgccca ggctggagtg 70440 cagtggcacg atcttggctt actgcaagct ccacctccca ggttcacgcc attctcctgc 70500 ctcagcctcc cgagtagctg ggactatagg cgcccgccac cacgcctggc caattttttg 70560 tatttttagt agagacaggg tgtcaccgtg ttagccagga tggtctcgat ctcctgacct 70620 cgtgatccac acacctcggc ctcccaaagt gctgggatta caggcgtgag ccaccgcgcc 70680 tagccctatt tcttcttctt taaattgttt ttattaaaaa aaaaaaaaaa ggccaggcgt 70740 ggtggctcat gcctataatc ccagcacttt gggaggccga ggcgggcaga tcatgaggtc 70800 aggagacaga gaccatcctg gttaacgtgg tgaaatcccg tctttactaa aaatacaaaa 70860 aattagctgg gcgtggtagc atgcgcctgt agtcccagct actcaggaga ctgaggcagg 70920 agaatcgctt taacccagga ggcagaggtt gcagtgagcc gagattgcac cactgcactc 70980 caacctgggc aacagagtga gactctgtct caataaagaa aaaaatgaga cagggtcttt 71040 ctgtgttgcc cagggtggtc tggaactcct gggctcaagc aatcctccca tctcaacctc 71100 cctgcccagc tgctgtgccc agcagtattt tctataatta gcatagtgta cttttcagat 71160 tatcttgaag acccagctct ccttgacctt gaaccttccc tgaggcctcc agaggcaatt 71220 tcctcctcag gactcagctg cccttcactc ttccctcgtg cacagcctgt cctgtgagta 71280 ggtgcaggta ctgactggtc ttatctttcg ccctgaaaat ataaacgttt taaaggcacc 71340 tgaggatttg tcagcacctc cccaccccca ccctgggtct gacctaggac tctgagtcag 71400 ggaggcctcc ctccactgag taatagtagc cttgggatct gctcagcaag aggattgtat 71460 agtttaaggg aggaagaagc accagtgctg cctccccatg ggaaaaatga agccccccct 71520 cccgcaacca ccaccaccac agaaccacca accccaacgt cacttggcag attagcaaaa 71580 gaactagcca gacgctgcta ggcccctcag gcttctagat tcgctctcca ccacatttct 71640 accaatttat gctcatcttg ataagctcct cctttgaagg gtctggaatg gtctggagtg 71700 gtctggaaag cagggtcaga tacccctgga aaactgaagc ccgtggagca gtgatctcta 71760 caggactgct tcaaggtgcg tggtgaacag acgtcttttt gcaattatgc cagttgctcc 71820 tctgatactt gagggttatg tgaatctaac aacagacaaa aatctctgcc tcctggggct 71880 tatattcttt gggaagagac aagcactaaa caaataagga aatatatata cactctgcca 71940 gatagccata agacggatgg agaaagttga agcagggaag ggagatggaa gtacctgagg 72000 gtggactacg catatttgaa atagggaagc cagggaagac ctcactgaga tggccacatt 72060 tgagaaagga cctgaagaag gtgaggcagt gagctgtgga ggtgggtaac tggggagggg 72120 gaagaacttt agctagagag aaaagccgct gcaaaggccc actgaaggca ggatatgcct 72180 gctgtgtcga gaccagcagg aggccgtgtg cctggtactt aatataccag aaggagtgga 72240 gcaggcaggg gtaccagttc cagaaggact tgctggcagg tattaagatg ggctttttct 72300 ctgtgatagg aagctccagg aaggatttga tcacaggagg gacagaatct gaattgtatt 72360 ttagaaggat cactttgact gctgtacaga gagtgggtgg tgaggtggga ggcagaagcc 72420 agaaagccag ttaggaggct gttaagtaat ccaggcagga gatggtgttg gctgggacca 72480 gggtcatagc agtagaggtg gtgagaagta attagattcc gtatatttat taaaggcaaa 72540 gccagcaaga ttccttgact ctaacttaaa ggttggagtt gtcatttact gagatgagag 72600 agaccatggg gggaaggtaa agggctcggt tttggatgtg ttaggcttgt gctgccattg 72660 gacatccgtg tggaggtgtc agagaagctg gcctggagaa ataaattggg gaatcatcag 72720 catactgaag gcattggaag gcacaagctg gcataagatc agaaagagat ggagcgtaga 72780 tggagaatga aagagacctt tagagattag gaagaaaatt actgactgag accaacaaag 72840 tagattgata cggagcagcc agtgatatca gagcaaaacc tgggaggaag ccaaatgaag 72900 agaaggcgta gaggaggagg gatcgagcag tcatatcact atcacatgct atcgtgggtc 72960 aagtaaaata aagatcataa gtgttaccga aagtttcaaa gtacagtgac attggccagg 73020 cgcggtggct tatgtctgta atcccagcac tttgggaggc caaggtgggt ggatcacttg 73080 aggttatgag ttcaagatca gcctggccca catggtgaaa ccctgactct accaaaaaat 73140 ataaaaatta gccgggcgtg gtgacgcatg tctgtagtcc cagctatggg agtctgaggt 73200 gggagaattg cttgaatcca ggaggcagaa gttgcagtga gctgagatcg agccattgca 73260 ctccagcctg ggcaacagtg agaccctgtc ccccaaaaaa aaggccgggc gtagtggctc 73320 atgcctgtaa tcccagcact ttgggaggcc gaggtaggcg gataacttga ggtgggttca 73380 agactagcct ggccaacgtg gtgaaactcc atctctacta aaaatacaaa aattagctgg 73440 gtgtggtggc aggtgcctgt aatcccagct actctggagg ctgagacagg agaatcactt 73500 gaaccgggga ggcagagttt gcactgagct gagatcgtgc cactatactc cagcctggac 73560 gacaaagtga gactctgtct caaaaaaaaa aaaaaaaaaa acaaaggctg ggtgcagtgg 73620 ctcatgcctg taattccaac actttgagat gccaaggcgg gaggatcact tgtgatccgg 73680 gaggagttca gttcaagacc agcctggcca acatggtgaa aacccatctc tactaaaaat 73740 acaaaaatta gttgggtgtg gtggcatgaa tctgtagtcc cagctactct ggaggctgaa 73800 gtaggaggat cacttgagcc taggaggtgg ttcagtgaac tgaggtcaca cctgcacccc 73860 agcctagtga cagagcaaga ctctgtctca aaaaaatcac ctaccaccct cttcatacag 73920 gggaactttc ttctattctt tttctagtca ttttcatgag aatcatgatc tttatgtaaa 73980 ttcgtattgt atttttcact tatgttatta aatattctat gaaaacatga gtttaatgac 74040 taggtaatac tcagttgtct gggtgtgccc taatatattt aaccattcct ctattattgg 74100 gcattgggcc atttctgatt ttttgttatt ataaataaca ttgcagtgaa cagctcagtg 74160 ttttcctata aggggaagta tagggtcaaa gaacgtgaac gcttttaggt gagacttgaa 74220 cttgagatcc ctgggttctc ctggacagac cgaccaatca cagcttaact gtgggatttt 74280 tccatgtctc ctctctccct gggaaggttg agaagtctct ctgccctgca gatccctatt 74340 cctgcctccc cgctgcaatt gcacgtccca tttctcctgg gccagtcaga gtttcattgc 74400 ctctcatggg gcctgctggc cacccactca gtggctgccc ttctccttac caccagcctt 74460 cgtcccctca cctagagcat tcacaggcga gcaagagcca gtcagagctt ccctcagagt 74520 agggggtgta gtgataaatg aaatagccct gccttcactc atctcactgg gtcgattaag 74580 tgatgtgtga tgtacctgaa agcacttcag gaacttaaga gtctcatgct ctaccaactg 74640 agctagccag gtgcccttaa agggctgtcc tagtcttaat tgtttgtaaa gaaataggta 74700 gctgggcatg gtggctcaca cctgtaatct cagcactttg ggaggctgag gcgggtggat 74760 aacctgagcc caggagttcg agaccagcct gggcaacatg gcaaaacccc gtctctacaa 74820 aaaatacaaa aatttgccag gcgtggtggt gcacacctgt ggtcccagct actcaggagg 74880 ctgaagtggg agaatcacct gatcctagga aatcgaggct gcagtgagct gtgattgtgc 74940 cactgcactc tagcctgggt gacagggaga tcttgtctta aaaaaaaaaa aagaaaagaa 75000 aaaggtataa gtggtggtgt ccacattgta accacagagc ccctaaagca gggctgttga 75060 cctatggttg gcatcctggt cacaagagct ctgtgtcaga tgtgtgaggg gcttgggtgt 75120 agttgtcaag cctgggaatg tggctgttag tatcgattgg cctcatgcca agcagtgcca 75180 agcgccacgt ctgctcccca gatacctgtg tttgtatgtg tgtgtcatag tcgggaatca 75240 ctgagaccag gacctctggg aggctgaacc ctgggagggc agagcaggaa caggccttgg 75300 agatgatcta attctgacat tttactcagt gaggacacag gtggggcagc cagagaggta 75360 ctcagctctg agtcagggcc ccatccctca actcctcagc ctgagtcttc ccaggaacac 75420 aacaggcagc tgcttcccag accttgcagg gctccaggtg gccttcccct cctgccgact 75480 cccttgccag gtttgctgta actattgctc ctccgagcct ggctgaaatc atcttcagtc 75540 tgttatcacc ttcagtctgt tctcatcttc agtctgttct ccacagcagc ctgaaaagtc 75600 tttgtaaaaa tgcagctctg ttcctcccca tcctcttcat aacccccttg gctctctgct 75660 cctcccagaa taaataccca catcctcagc ttacccttca agacgctgag tgacctctag 75720 ccacagccct tctctctacc cacagacagc ccaggtcctc attccttggg ccttttcaca 75780 tcactgttcc catcaccctg gaatcccact cagcctgcct gggccagagg ctacgtaagg 75840 tcctcaggaa agcgtctcct ggccactcgc acctgactca tgtgctctga gtgccacacc 75900 ttgcttggca tttgtccttc atagccctgg tcattgctgg ctaatgtagc ctggcacatg 75960 gtggtaggta cttgacaaat atttgttgaa caagtgaatg aaaccagctg gcagggacca 76020 gaagggcaga ggggcaggga cagtgtccac aaccatagcc cctgtggtct tagctgagaa 76080 gggctctggc tcggtctctg gtgagtctct ctgctggccg gcagaaccac cctaccctgg 76140 aagccctctc catgtagtgt gaggggtctc aggcccattc aggcttcagt aggagagatc 76200 tgggtctctt ctgggacctg agcaggcctt tcctttacct cgttggcatc tctcagcatt 76260 ttatttggtt ttaaaaatgt ctcccccttc ccccgaccaa ccccagatta taaaggtatc 76320 ccacgctcac tgtgaagaaa ttggataaca taggaaaata taattaagaa aatcgaaatc 76380 acctacagct ccacccctca gagaaaagta cccttaacat ttgtggccat gtcttttcag 76440 ttttgttttt atgcatgtgt atgtgctttg acattgctga gcttctcttt gtgaaaacca 76500 cagatttttc gcagtcttca gtggctgcgg tcagaggtgg tcagcacgtt agtgctccca 76560 gtggagaggt gatgcaggga tctgggtgcc cctttctccc tactgcaggt gggtgggtgg 76620 gcacacccaa ggcccccttc tgtggcagct ttcctatccc tggtgaagag taccctcctg 76680 tctctggtct cttctccagg acattctcca gccctggagg ctgttcctag ggggctgcct 76740 cttggggcaa tgggaagaat tgtggaatgc gtgggtggcc tcctggctgt cttttctctg 76800 gttccagcac tctctgaaca aaaccagccc caggccactc ctggggattg gcctcaggtg 76860 actctctggg ctgactctgg ccagctagaa ggccacccgg gcttctcctg tgtccttaag 76920 agggtggggt gacctggatt ggcaaatagt aagcagagaa cacggcagag ttgctggcct 76980 agaaaaccag gtcactaggt cagcacttgg attaatgatt gataagaaag gttgggagag 77040 aatcagccag acccattctc ccatgacatc cctcacttct gatcccaaaa gcacctgcca 77100 cggagacggg gcaacctctg tcttgtatcc agaggctgcc ccttattccg ttgcagtatg 77160 ggaggaagga ggtgacagca actgggtcac ctgcatgtgg ggcgagaagg ggaggaaggt 77220 ttaggccctt ctccctgaaa aaagaagcat gtgcccagag tagggggctc cctgccggtt 77280 ctctccccac aattgatggt gaggcaggga actgggaagt tctgtctgtg gaggtccctt 77340 gggtttcagc aggctagcac atttctagga ctggaatatc accatacaat cccttaacac 77400 aagccatggg tgataataat agcagcagcc actgtggtcc ttatttgtag gacccttact 77460 atctacctgg cactgttaag cagttactgc atcgttattt attccacaca gcagcctctc 77520 aggttaggtg gtagtattcc agttttaatg tcttccccaa gtcataggat gggagggatg 77580 agccagggtg tgaacccagg cctgactgaa gccggtggaa ttcacttctt tggagtattg 77640 catctcgatg gcagtaatat atgaaaactg atacagtggc aagtgctaca actcagaata 77700 gtgtggctaa ggctcatgtt tttagtatat ttattcatcc agcaaacgta tgttgaggac 77760 ccactaaatg ccaggcaccg tgccaggtgc tgggacctac ctcagtaaga tacagggcat 77820 tctctgggaa actgataaca tggtgaggcc agagcagtta gcagacattg cagtttggtg 77880 tagtgattgc agatctggag ggcccgggtc agggttgtgg tgagaaggaa cagaatggag 77940 ctagtccagg agagcttccc ggaagaggag gtgcagacag gtcgcgaagg agagaaggtt 78000 ttagctagtg gaggggatgg cattcaaggc aggtgccagt tcaagcctcc atgctcaggg 78060 agtgatgtgg gatcatgagg cacataggca ggcagagcag gaggataggc ctggagaggg 78120 aggcagggac gagagcctgt ggctgtcttc agcacacagg agtcattttc tggcagtaat 78180 gtccagaagg gctttgacca aggtgttctt gacagaaaca gctggggact ggcagccaga 78240 cagaggattc agaggtgatg gctccagact gagactaagg agccccagct gggcaaggac 78300 atgccagctc ctggtccagt cttacccagc accatatatg aggcacacag tagagtggct 78360 aagacttgag ccaacctgcc ttgactcaaa tcctgcttct ctagttactg tgtgaccttg 78420 agccaattac tttgcctctc tgtgccataa tttccacgtc agttaaatgg agatgataat 78480 agtacctgcc tcttagggtt gttgtgagga ttacatttgc caacatacac agagcatggt 78540 gtgtaataag tccttggaga agggaagcta gtactattgt gatggaggtg gtgatccagc 78600 ttaggctctg gtacagtaag aaggtccatc aggctgtacc atgtggcaga tggagcccat 78660 ggaggatgga ggcccctggc acactgactg tccccactca tcccctgggt agggcagaag 78720 tgttcccaca ccgtacagcc tgccagtggg catcagggaa acacctgagt caccacgagg 78780 agtacgaggc acttgggggt tgcccagctg accaggagca cagagggctg ccctgaaagt 78840 gagcccctcc ctactcctag cagagacggg gaagggagac aactggactt ggtgggaagg 78900 actgggcccc aggtggctta gttgctctgg ccccagacca ggtctctcta gagctctggg 78960 ccatttgggt taagcttcta actaccactg cccatgaaat tcacctgcca atgggcaatg 79020 gatggagtca tgcccctccc tgcacctaag ttctctgaag ggctgagttt cttcttgggg 79080 ccagacagtg ggtattggaa tgggagcatg ggggctgcat gtgcactgca ggctttgtgt 79140 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtacacacat acatatatgt 79200 cagaattgga atagtccagg ggtttgtttt gcaagaaatc cttttatttt tattttttga 79260 gatggagtct tgctctgttg ccaggctaga gtgcagtggt gtgattttgg ctcactgcaa 79320 cctctgtctc cctagttcaa gcgattctct tgcctcagcc tcccaggtag ctgggattac 79380 aggtgcgtgc caccacaccc ggataaccag gggttttgcc atgttggcca gggtagtctt 79440 gaactcctga cctcaggtga tctgcctgcc tcagcctccc aaagtgctgg gattataggt 79500 gtgagccccc ttgcccttcc cccatgcccc atgcaagaaa ttattttgct agggactact 79560 ttttggtgag gggctacatt gctgcattcg tgccccctca ttctggctca gggcccttgt 79620 gttgtctggg ggtctgaagc tccagcctct taagtggggc aaagctgtga catgatcaga 79680 gggcttctgg ctctggcata gtgctggggc gtggagaggg gagatagcca acactgggag 79740 ccagaccaaa aaaaaaaaaa gccagggttt tttgccggca tacaaattaa acttagtgcc 79800 tggctgtatt gtggttgttg ttgttgtttt gagacagagt ctcctctgtc acccaggctg 79860 gagtgcagtg gtgcgatctc ggcccactgc aacctctgtc tcccaggttc aggcgattct 79920 cgtgcctcag ccacccacgt agctgggatt acaggcgtgc gccaccatgc ctggctaatt 79980 tttgtatttt tagtagagat ggggtttcac catgttggcc aggctggtct cgaacacctg 80040 acctcaagtg atccacccac cttggcctcc caaagtgctg ggattacagg catgaaccac 80100 cgcacctgga cttttttttt tgtttttgag acagggtctc aatctgtcct caggctggag 80160 tgcagcagtg cgatcttggc tcactgaaac ctctcccctc cgggttcaag tgattctcgt 80220 gcctcagcct ccgagtagct gggattacaa atgtgcacca ccgcacctat ctgatttttg 80280 tgtttttagt agagacaggg ttttgccatg ttgcccaggc tggtctcgaa ctcctagcct 80340 catgtgatct gcctgcctca gcctctcaaa gtgctgggat tacaggcatg agccatgccc 80400 agcccagcct gtgctatctt tagtgccagg gcttaagaaa ggaagaggag aatagggttc 80460 attgcaggaa ttgtggtggg tggggtggca gctacctgag aaaatctaga cagtgggcct 80520 aggtccagtg ggtcagggga gagagcctag gggtcaaggt tgctcctggc tgggcaagct 80580 gagaacaggg agcaaccttg gctcagggta gatgacagac ctcagctgta gatggagcaa 80640 ggaggccata tgaaaatggc aggcttctgg caaaatgcag ggatgggctg cagcacagca 80700 gttcaatcta aataggagca gcccaaggga cgtgggtagg ttttagaaca gccaggctac 80760 agctcccctt cctcttcact ggtgtccttg agaggccaca gtcagaagag acaattccag 80820 gctagggaga gatgtcagcg tgggaactag agaccctggt cccaaattca gaactctagc 80880 cagagtccag aactgcatct gagtctaaga ggcaaccact tacttacacc tcccgaaact 80940 gagaacagtt ttaccggttc tggtgaaaaa aacccactac cagctcatat gtgggagtgg 81000 gagctgaggc tctgctgagt aacacggccc agtgcacaca gctgtcaggg cctggcccgg 81060 ctcgagtcct gctgtgtccg aagctctgcc gttttctgct tcgtcctcta gcatgttctg 81120 agatgccctg ttgggatcct caggcctgtc acttttgtca tctgcaaggg gggcctataa 81180 ggccttaatg ggggctggcg aggcttgagt gggagcgtga cgcatagtgg gagctcagtc 81240 agctccctcc cctccctcca ggatttggtc ctgacaggga gctccatcag gtcttcccca 81300 gggtcaccag aggctcagat gctgtgttgt caagtatata taccaggcag tattgtagct 81360 tttcctcctc tgttactgcc tgctaagcgg ggaccccaca tcttcacgtc ctcctcttcc 81420 tggctctcat caccacagcc tggtgtgcat tgccccgtgt ccactgacac ccacgccaca 81480 tgggtccaaa aacagagtgt gttctcattg cctcatccca ctcaccctgg gctctgccgc 81540 cactgcgctg tggctctcgg gggtcccacc atctgaccct tgtcacccgt gctgctcctt 81600 ctcttgggac tcatcccttc tgaggcctcc cgtggcccct tctcaccctc ccctacctcc 81660 tccatctcca ggctttcaat ctggtggagc ataaagaatc agtgtttctg gtacttgtta 81720 tttggataat ggcacctaat gctaaatgac gagttaatgg gtgcagcaca ccagcatcgc 81780 acatgtatat ataggtaact aacctgcaca ttgtgcacat gtaccctaaa acttaaagta 81840 taataataat aaaataaata aataaataaa ataaaataaa ataaactttg aaaaaaaaga 81900 tttcagctaa aattagtttc ctctccatgc cgagtctgaa tatattataa aaataaaaga 81960 ataattgtct ttcacacaat taaaaaaaaa aaaagctgac taatgccact acagtcacac 82020 actgctgttg gtaattgcta gggtcctttt tccagtattg tggccaactt ccaccccaga 82080 accccactct actcttgagc taagtgtctt tatgtctcct attttaaatt aggattgcgt 82140 tcagttgcat gcaacagaaa tccggcaatc agtgcctggt aaaacaagtt tgattttttc 82200 acctaacaag cagtctggag gccggcattg cagggtgggt ccagcagctt tgggaggcca 82260 cagtggccag gtgctttcgg acttttcacc caccctccta ccctgtcttc tgctgctccg 82320 aaggcagctg ctacacctct aggcatagta ttcctgttcc aggcaggaag atgggagggg 82380 cagaagtttg tctctctatt aggaaataat agctttttcg gaagctcctc ctggtagatt 82440 tctgttcaca tattgctggt cagaattatg ccaatgaaca ctgtagctgc aagagagctt 82500 ggggaaattg agtgtttagg taggcacaga ttgctgatcc agagaagact gagactcaga 82560 aggaaggaga atggagctgg ggagaagtag gagtgactgc cccagctgcc acttggagaa 82620 agccacgggg tgagcaggac agcacctgtg aacatttcct gtgctctggg tctggtgctt 82680 ctcaagttta acacccaggg gcgagtggtc agggactgat ccttgaggaa ctgatgtaca 82740 ttaaacaaac tatcaagtcc ctgcaatgca gtgtaaatga gtaaagagcc tgcaggggag 82800 aaactgccac tagtggagac catgcgagtg ggcagccgat tgcttggctc tgggggtggg 82860 tgccagaggc atgcaggtgc ccaagggcca ggtctgccca attttcaaga gaagccagaa 82920 aaccaaaatt tttaaaggtt ttttcgttct aactctattt tgaaaaaagt tcggtgttac 82980 agagtaagtt gcagagattg tacagagtcc ccacatatcc tttacccagc tcacattaaa 83040 tcttacataa aggaacactg gtacaatact attaaccaaa gtcagtgtgt tgttcagacc 83100 ttaccagttt ttctgccact gccccctttc tgcttcgggg atctggtcca ggatcccaca 83160 ttcatttagt tgtgtctctg tggtctgctc ctgtctgtga ggcttcctca gtcacaaagc 83220 tagaggttta cgtgaccttt tccaggtttt cccactctaa tgatttggaa atcatacatc 83280 ggcatcttga gacatctttt gagaagtaaa gggacttgag ctgattgaat gttgatcact 83340 ccctctcatc ttccacatta ttaagttttg ccttcttatt ttattataat ttaaattatt 83400 tttaagccag gcacagtggc tcatgcctgt aattccaata ccttggtagg ccaaggtggg 83460 cagattgctt gagcccagga gtccaaaacc agccggggca acatagcaaa accttatccc 83520 taaaaaaaaa tacagaaatt agctggtgtg gtagtgcaca cctgtggtcc cagctacttg 83580 ggtggctgag gtgggaggat cccttgagca cgggagataa aggctgcagt gagccatgat 83640 tgcgccactg cactccagcc taggcgacag agtgagactg tctcaaaaaa caataacaac 83700 aacaacaaaa aaacccatca gaaaacagct aagagttttg tggtggtgtt ttattttagg 83760 ttcagggtac agatgcaggt ttgttatgtg ggtgaacttg tgtcacaggc gtttattata 83820 cagattattt catcacccag gtactaagac tagtacccga tagttatttt ttctgctcct 83880 ctcctcctcc cagcctccac cctcaagtag gccccgaggt tattgttaag tccaagcaag 83940 ggaaggcctg gtggaaatga atgtgtagcc ttaacatgtt ttgtacaaac ttggctgtac 84000 attacaatca ccaggacaat taaaaaataa gaaacccaat gtccaagctg ctccccaaac 84060 caatcaaagg agaatctctg gggatgggac caggcatctg tctttttaaa aactccccag 84120 tgactacagt gtacagccga gttggcaggt ctagagcagg gagagagagc agaaggagtc 84180 tctgatcact ccaggttctg gttctgatga gtgggtagac aggcaggtgg gttgctaaag 84240 taaagattac tagaggacga atgggtttgg ggaaaatgat gggttctgtt tggatagatt 84300 gagtttgagc tgtcggtaaa atatctgaga gttggggtcc cacagatgct taacatctta 84360 aagcagatct tttcttctcc tgatccttcc acaaggtctg gtgcagacat ttccccgctg 84420 ggagactctc cacagtacct catgatctta tcatctctga ctgtggcatt tgtggataat 84480 gttttacttg ctaagggttg gaactgtctc ctcctgggtg atgtggacgt ggcatcacca 84540 cccagtaagt tgtcaccttg gaaagactcc aggatggagc acatcttcct ttttctgatg 84600 tctctgctat tgaggccatg gttgcagatc tgctgtcaac atgagccaag aacagcaggg 84660 ctttatgtct tttttccaca cccaaccact gaaaggatgg gactcacccc ttcaggaaca 84720 gtagcacagc agagccgtga ttcttgggcc tgagtgctga tagggacagg gtcggggatg 84780 ggaggcgtga tgcatatctg gatctccagg tgggctggag agttgggaaa agcccctcaa 84840 gtcattccaa tgtgcttcct ggagactcag aacagaaggc agcagaggag gcagggaagg 84900 gtggcacagt gatgtgtcac ctctctagtt tatggagcct gctccgaacc ccacaaaccc 84960 actacaagag ccaggtggga agggtgatag ggcaggtgac ttgtgataca aggagcagag 85020 accatggtat agcactgcag gaactaggga aggagcagga gcagaagaac cccctccatg 85080 acagcagccc ccaagcgtgc cccactcgca ccatcctctt ccttgtcact gcctcccctg 85140 acctcttcct gtcttctcct ctgcccaggc tgatgggaac caccctgtag aggtccatct 85200 gcgttcagac ccagacgatg ccagagctat gactgggcct gcaggtgtgg cgccgagggg 85260 agatcagcca tggagcagcc acaggaggaa gcccctgagg tccgggaaga ggaggagaaa 85320 gaggaagtgg cagaggcaga aggagcccca gagctcaatg ggggaccaca gcatgcactt 85380 ccttccagca gctacacagg tgaggagagg actggcaggg gacacggggc agaggaggca 85440 cagcccagtg cagtggggat cctggccctc tgcaaacgcc atcatgtggg gcgcagagta 85500 gcagagtgct gaaggactgg agccggaagc ctgggttcac gcccagtgcg gtggggatct 85560 gctctgccac tcaccagcag tggggcctgg agcaagctgc accacttcct tctgtaaaac 85620 aggccaaagg atggtaggtg atgtggatat gggctttcgt gagaattaaa tgagtgggca 85680 tctgtaacac atgtcatcat cattattagt attcccacca ctgttaacac aaggcatctg 85740 agaccaatct ccactaacag cacactagaa agatcagctg tacctgggat tgttatgatc 85800 agtcgaaaca cacagttcaa ttggtctggt aataattata cacatcatca ttgagccgta 85860 tcataattag ataaaaatgc taagcagtta gtatgaataa ctcaactaat attattttta 85920 tgcagccaag cagttgtagc ttctcctgtc tcacttattg gttccccttt atttatcagc 85980 agtgcagaca gttttctcta gtaccctgta ctgttctgtc atcttcatca tcatcatgtt 86040 atgtttacct tcaaaagcac ccagaatcca tgcagcccct ggctttccct gctgcagttc 86100 cccctgcttt tttgtttttg tggtttttga gacaaagtct ccttctgttg cccagactag 86160 agtgcagtgg cacagtctca gctcactgca acctctgcct cctgggttca agtgattctc 86220 ctgcctcagc ctcccgagta gctgggatta taggcgtgca ccaccacgcc cggctaattt 86280 ttgtattttt ttagtaaaga tggggtttca ccatgttggc caggctggtc tcgaaccgct 86340 gggctcaagc aatccaccca cctcagcctc ccaaagtgct gggattacaa gcaggagcca 86400 ccgcgcctgg cctgatccct ctgcttcttg ctttctccca ccacttctct ccttacccaa 86460 gtgctgcagg cctgcacatg cagctcatcc tcgctcctcc ccttagaggg aactcagggt 86520 cataaggata tactgctggg agtagacacc ctgcactcgc tgctgctttc agctgcagga 86580 gatcctagcc acaggcggtg actcaggcag aggagcaagc ccaccccgca gaaatgggct 86640 ctggacccct gtgactgcat ggagcaggag gaggcagtta gtgtccaagc taaggcaggg 86700 tgaaggctgt ggaggtagcg caggctcctg tttgggtctg cagctaagat gggtctgaat 86760 cctggagcca caccaccttg aacctgcaaa gcaagtccca gaattttgca gaagttcttc 86820 ctcttcagtg tatagagttg ggtggaggga gtatagagcc atcattcagt ccttctcatc 86880 tcacccagga tcacctggga gtgaccaaag taggccaaat gtgattctag ggccaaagtg 86940 aaggtgtccg ccattccccg agtaagcaac ttcctgtcta ttggctggta actgggatgg 87000 ctttggagac acagcctgtc tcagaagaga ggaacagcta accaggagtg ataattcact 87060 cagtgccagt agcacatact gttctaaatc tgaagaaggt agggagggtt agaaagcaaa 87120 gggaagacag tggggactaa gaatgagtgt gggatgtcat gaggattcag gggagaatgc 87180 aaggagaaga gacggccaca gggtttgtgg ctatgccgcc tgccgtggcc cacctcatcc 87240 ccacatctga gcccaggcag atggaagtcc cctgcagagc agtgttctgt cctggagttt 87300 cctccacacc ccttcttggg accagggctg tttcccaaat agcatcactg ttcccgtgca 87360 ggccaggtga ggtgcctggg agcaggggta gggtgaggca ggcatggcgg tgacctctca 87420 ggcagcttcg ttgtgggatt tagggcacag cgggaaacgt ccagggcaga ggttgcctca 87480 ccagtcacat ggtactatgt aggcggtggt gtttaataga ttctgtttat caataagtgg 87540 atgaagtgga gatagacttc cttgttcagt cattgaaaca ggagagaggg ttacttgaca 87600 taatcacttt gtgttaaagc tgtagtgtat gaaaagttcc caaaaattct acaagacaag 87660 caacctagca ggaaaatcag aacaggtaac agtaagacaa atacgaatgg cctgaaatcc 87720 cgacaggttt ttcagtctaa ccaactcaaa gaaagcacct taagttgcaa tgccatttca 87780 cctgtcagat gggtggggag gacttggtcc tcccaccacc aggggtgaaa gggtagtgtg 87840 ggccaacact tcttaatgtg tgattctttt tttttttttt tgagacagag cctggctctg 87900 tcgctcaggc tggagtgcag tggcacacga tctcaactca ctgaaacctc tgcctcccgg 87960 gctcaagcga ttctcctgcc tcagcctccc aagtagctgg aattacaggc gtgcaccacc 88020 gtgcctggct aatttttgta tttttagtag agatggggtt ccactgtgtt ggccaggcta 88080 gtcacgaact cctcaggtga tcctcccgcc tcggcctccc aaagtgttgg gatttcaggt 88140 gtgagccacc acacctggcc aagtgtgcat ttcttttacc ctgcattccc acttctagga 88200 acgtgtcctg tgtaagtact tgctcagcat gctcagagat tcccatgcca gcttatttac 88260 tgcaggactg ctcataagag caaaaatagg aacgtgccca tctgtgggga actggttatg 88320 taaactgtgg tagagccata tggtggaata ctctggagtc attgaaaagg ataaggctgg 88380 tttatatata tattaaataa gttcctgaca agttggggat cccattgttt aaaatcctta 88440 tgtttcatag aaggcaccgg aagagtctat accgaaggac tcaggggcta actctgagga 88500 gcaggatcat ggttaaggag gctgtgtcat aagatcctgc atacccttct gtgtttttct 88560 caacaagtct gcactacttc cataatttga aaggcaactg tacttttttt aagggtaaaa 88620 ttaactgttt aaaagggggg tgaggatctg gcaggtgagg ggccctggag acagagcctg 88680 atcatgtgtc cagagcagaa gggcttggag atcacagaga ccagggcagg catcccaggt 88740 ctgtcactgt gagctgtagc cctcgccagt gccatcactt ctccaagtct cattctcttc 88800 acctttatag gctgtgtcct acccagcctg agggccagcc aggtgagagt cagtgggggt 88860 atgtgacaca tcgcaggtct cagcacctgc tagttctctg ccccatcccc atgagttatt 88920 actttggcct cgggagtccc ctgagggacc cataaggaca gcgcacagct ttgagccctg 88980 aaacagcagg aaggctccac agtcacccct gaccaccctc ctcagcagtt cagcactgac 89040 tctcagtgca gatattgagc tcaacacctg ccatgcacct tctcaccctc cccccagcca 89100 ggcaggacag accttcccat caacaaggaa actgaggtta ccagaaaccc acttgccagg 89160 gtcccctggg cgtgtaggag tagagacagc tagagccagg attcacctcc cattcccatt 89220 ctcatccctc ctgcttcagt gacatccatc ccaatcaccc ttgagaaaga ggaagataaa 89280 gtgtccttcc tcctggcctc agcacctcct cacttggcaa gcacttggtg ggcatcaggc 89340 tgggcccagc actgtgccca gggctggcca tacagtggtg gacaaggctg agtgcctgtc 89400 ccaaggagcc cacagttcag gcaaacagga cagcagaagt aaagaggctg ctcagtaacc 89460 cagcctcaaa tccctgcaga atttggcctg acttcagaag ctgctgggtg cttgtttgcc 89520 tgtgagctct gccagcaaag taaatagacc aatcgggcgc ctcctggctg tcttcacagc 89580 catgacaacg gcattctccc agcattggag gctagatatg caggagagct ctcagccaca 89640 aatgatgtga ataaggtcaa tagtgtctat ttgctgaact tgtcatcaaa gatggtgaaa 89700 ctgatacttt actctctggg tgagagggct agtgtttaca ctgaatccaa acatttggat 89760 aatgacatgg tgcctcctca gcagtgactg tgtcttcttg ttcatcataa taatgttatt 89820 gattagcggt actgaacatg taggtatgcg tagtagtgag atgccgaggt taagagctca 89880 gctccaggaa ttgcatggtt ctaggttata attcttgctc tgtcactgta ttagtcaggg 89940 ttctccagag aaggagaacc tgtgtgtgtg tgtgtgtgtg tgtgttgaaa gaaagagatg 90000 tgttttaacg aattggttca cacagttgtt ggagtggcaa gtccaaattt tgaaaggcag 90060 gcaggctgga gagccgggga agttgatgtt gcagctcaag tccaaaggca gcctggaggc 90120 agaatttcct gttccacagt ggggccacgg taggcactca gtcttttttc ttaaggcctt 90180 cagttgttgg atgagggcca ctcacattat ggagggtaat ctgctttact caaagtcttc 90240 tgatttaaat gttaattgca tctaaaaaat atcttcttag tggccgggca cagtggctca 90300 tgcctgtaat cccagcactt tgggaggcca aggtgggtgg atcacctgag gtcaggaatt 90360 caagaccagc ccagccaaca tggtgaaacc ccatctctac taaaaatacc aaaaaaaact 90420 agctgggcgc ctgtaatccc agctactcgg ggggctgagg caggagaatc gcttgaaccc 90480 gggaggcaga agttgcagtg agctgagatc gcgccattgc actccagcct ggggaacaaa 90540 agcgagactt catctcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aacaccttct 90600 tagcacattg tttggtcaaa caactgggca ccgtggccta atcaagttga tatgtaagac 90660 caacatagcc gctgccagca agttactaac acttctgagc cttggagcct cggcttcctt 90720 gcctgtaaag tgggggtaag aagaccatct tgaaaggttg ttttgaggtt ttacgtgaga 90780 tgaaatgggt gtccaatgga gtgcctttag caggacacct gcttaccccc acctctaccc 90840 aggacatcat gatgcggggg aagtggtgga accttcagaa cccagcccct agtatgagaa 90900 gaattctgcc atcacctttc tttcagcaga gagggaagca gtccccttcc gtgacagtca 90960 catgcgtgag tgatggctcc ttccaatatc ctgcacttct tactgcccct tacctggcgt 91020 tgctccactc tgtctcatgt tctagttttc ttatctcttt ctcttttttt ttctatttta 91080 tttttttcct gtaggcacaa gtgagttaca gtaggttaag ttacacctta cctttccttg 91140 tttttcttgt ttatttattt gtttatttat ttttttgaga caaggtcttg ttctgtcacc 91200 acaggctgga gtacagtggt gcaatcacag ctcactgcaa cctggacctc ccaggctcaa 91260 gtcatcttcc cacattcagg caatcctccc acctcagcct cccaagtatc tgggactaca 91320 ggcatgtgcc accacaccca gctagttttt aaaatttttt atagagatag agacagggtc 91380 tcactatgtt gcccaggctg gtctcaaact cctgggctca agtgatcctc ccgcctcagc 91440 ctcccaaagt gttgggatca caggtgtgag ccactgcacc tggccttttg ttttttttaa 91500 gtagagacag tcttgcttta tcacccaggc tggtatacga tggcacaatc acagctctcc 91560 taggctcaag gattttcctg cctcagcctc ctcagtagct ggcactatag gcaggtgcca 91620 cgacacccaa ctattttttt ttttttactt tttatagaga cagggtctca ctatgttgct 91680 cctggcctca ggtaatcctc ctgccttggc ctcccaaagt actgcgatta ctggcgtgag 91740 ccaccacact cagccgagtg caatggcgcg atctcagctc actgcagcct ccgcccccct 91800 gcccccgggt tcaagtgatt ctccttcctt agcctcccga gtagctggga ctacaggcat 91860 gtgccaccac gcccagctaa tttttgtatt tttagtagag atggggtttc accatgttgg 91920 ccaggatggt ttcaatctct tgacctcgtg atccgcccgc gtcggcctcc caaagtgctg 91980 ggattacagg cgtgagccac cacacccagc cctgctgtta cctctttctt acctcagcta 92040 tttgaggata gagtctaggt cctgtcctgc atatagaagg tgcttatcat gtgtcttttg 92100 agtgaacaca gagggtgaca ttgtcacgtg cagtgccaac agaaaccaaa tgactagaca 92160 cctgctgacc ttccatggcc acagggagga tgagaaaggc acctccagga ggtgtgaata 92220 atctgagggt attggccact gtggacaaga ctcggtggtg gaagtgagct cagagatcca 92280 aagggaacgt gagtagagac atgcagagtc tcgcagagga agcagacgtg caggccacaa 92340 agacaaaatt acaagaaatg ccccaacacg agcatatgct cacggactgc acagattcaa 92400 ggaggaagtg ccagaaaccc tgcctgcccc agcatctgca caggcatcca caaagggttt 92460 ctgtcatgtt atctcttttt cttccaacat ccggtgctgt gagttttatt gtccctgtta 92520 taaatgcctc caggagtggc aggtcctgcc cagccccatc agccagttgg tgctgcagtc 92580 aggacaccca ggcagcctga ggactgccag atgtgcagag agctatcgcg tgcctgctgc 92640 gtgcccagca cctcacacag ctctcagtgt ctgaccacgc tggtgccctg gtggacaaag 92700 ttcctgccct tgtggccctt ttgtctagtg ggtggcttcc aacaatgaac acacacagga 92760 tatgtcaggc ggtgacaaag gctactgaga agaccaaagc ggcttcaccc tcccagagca 92820 cagcttttcc ctcccaggcc cgagagatgt agggagccca ctgtcccagt ctgggtcaag 92880 aggaacagtt acaacacctt ggaatgactg aagttgggag ctaaggtgcc aggacaagac 92940 aggccatttt aggaaccaga gacttgtgtt acccctaaac ctcaccagcc aggaggggag 93000 cagcccactg ggtcctctct gccacagttg ggtgcttctt gtgctccccc tcctgtggtc 93060 aggatagggg aagcaggctt tcctgctgcc actcctagga cccaagggga agctggggga 93120 gtggggagac catgtgggaa ggatggagtg cactttgatt ggtagcaggt ggataggtac 93180 tcccatggtg gggaaatcaa agttgcttgg cctgtgtagt agacaggtat taccacaggc 93240 ctggaaaaca ttagcctggg tttatctctg tggcttcggt cccccaacat ccccttctac 93300 tcaaaatggc tgattctctg gtttggaggt ttggcacctg cttttgggga ggttgctcag 93360 tcattagaga agagtgcaga ggttggacgg tcatcatggc agaggtagga cagctggtat 93420 gtggggccat gaaaccagct ttgggctagg agtcaggaag ctgagactca gttcaggtcc 93480 cctttccaag atgaagagat catttccgag tgtccaaagg gcactgggaa gagaatccta 93540 gcagttctta tgaaatcatc tgacagcaga tatatcctga agtcatagcc ttagcaaact 93600 gcctttctcg ccaacacact caacaccccg tggttcaaac atctatacgt ctgatagaaa 93660 aacttggaga aattcagtgt gttgcttaat caatatgact gatacctaag ctgttctact 93720 caagaagttt tcatgggaat gaacttgagc ttcttgaaga gattgtttac agcatccctg 93780 gagtctatgc aagaatagaa cttcctgtgg gggatggtgg ggaggctagc tctgcagagg 93840 tctacagtcc tgcagtttct gcaaaacacg ttgcacacat ctccatatgg gccacacggt 93900 ggcaacctct gaaccaggag agctgcttcc taagggctaa gaccctctgt ctgctttctc 93960 tctcttgctt tcctcccaaa acacagccat cctcctcccc actgctaccc catggtgagt 94020 tcagcccagg aagcctctga cccagccctg atggtaatag ctgctgtgta ctggacatct 94080 tctgtatgtt aggctctggg agaaatgctg tggagacttt ttgcccatcc tcccagagaa 94140 gctgtcagta ccccctgttc taggtgaaga aactggtcct cagaaaggtt gactccctga 94200 ggccacacag ctgttaagtg gctgaggcga gggctgtggg gctgttccca cgccctggct 94260 tccaggcctg gcagcatgtg gagctgcccc tccatcgtgt gtccgcagac ctctcccgga 94320 gctcctcgcc accctcactg ctggaccaac tgcagatggg ctgtgacggg gcctcatgcg 94380 gcagcctcaa catggagtgc cgggtgtgcg gggacaaggc atcgggcttc cactacggtg 94440 ttcatgcatg tgaggggtgc aaggtacgga ctggggggag cggtggctgg ccactgaggc 94500 tgtggtcaca tggtgaattg accctccaca aagctttcct tgccttgggg tggggccctg 94560 actgtaccta ttgcagaatt cctgcccagg aggcagccag gcccttgtgc tccagacctc 94620 agctctgggc actgcaggag aagagggggt tcccttgtca agcctaccct gctgggcacc 94680 tgtgttgacc cagctctgtg ggtgattgtg tccccctggg gaccaggcag ctagggcaga 94740 atgagggtct tgggctgagc catcgtctta cctgaggtct gggtttcttg tttctcaagg 94800 ctcaaagcgg gctctgagaa aaaaggacag acatttgttt ttccagtgat atttgtgggt 94860 ccttcctaac ttcacaccca tcaaaatctc acagattatt ttattgaatc accacctcac 94920 aaatacacag aaccagatac atttatgaag aaatcaagtt agccgtgaga caatcttatg 94980 ttaccaaact aacaaagaag cttttttttt tttttttttt ttttttgaga tggaatctcg 95040 atctgtcgcc caggctggag tgcagtggcg tgatctcagt taactgcaac ctccacctcc 95100 cgggttcagg agattctcct gcctgagcct ccagagtagc tgggattaca ggtacatgcc 95160 accatgcctg gctaattttt gtatttttag tagagatggg gtttcaccac attggccagg 95220 ctggtctcga actcctgacc tcaggtgatc cacctgcctt ggcctcccaa agtgctagga 95280 ttacaggtgt gaaccactgt gcctgaccca atttttgtag ttttaatagg gacggggttt 95340 caccatgttg gctaggctgg tcttgaactc ctgacctcag gtgatccacc cacctcggcc 95400 tcccaaagtg ctgggattac aggcgtgagc cactgtgtcc agccccaaca aagaagcttt 95460 aggacttctt tagacattta aaatagttgt gttttttttt tgttttgttt tgttttattt 95520 tgttttgttt tgttttgaga tggagtttca ctcttgttgt ccaggctgga gtgtaatggc 95580 gcgatcttgg ctgactgcaa cctccacctc ccgggttcaa gcgattcccc tgcctcagcc 95640 tcctgagtag ctgggattac aggggtgtgc cactacaccc agctaatttt tctgtatttt 95700 tagtagagac ggggtttcac catgttggcc aggctggtct caaactcctg acctcaggtg 95760 atccgcccac ctcggcctct caaagtgctg ggattacagg cgtgagccac tgtgcctgaa 95820 ctaaaatagt tttaattaaa gttttagaat tactttcctt ctccatctcc cctccaaaaa 95880 aatttcttct cgttacttcc aaatgtcagg agtgtttaca ccttatacat aatcgggccc 95940 tttggcacag cctccctccc acctcctggt ggcctttcct cacctgttct tggtgcttca 96000 gggcttcttc cgtcgtacga tccgcatgaa gctggagtac gagaagtgtg agcgcagctg 96060 caagattcag aagaagaacc gcaacaagtg ccagtactgc cgcttccaga agtgcctggc 96120 actgggcatg tcacacaacg gtgagagctg accagggcaa ctcacgggct gctggctcca 96180 cacagcctga aaccaaggtc cagggagccc ttggggcagc ctagaggggg cacctgtaga 96240 gcagtgcctg gcgcacagta agcaccatgg ctgttggctg ttggctttgc tgatctggac 96300 cttggatctt agagcctaag acctgccact tcctcagact gccagcatct accatgggtc 96360 ccaggcgtgg ggtagtgttt acctccctat acctgaggtt tacgcattct ggcccagaag 96420 ggaactgata attgatacca gcttgaacaa tttggggaac accagtccca gaaactcaag 96480 cctgtctcag atgacctgac acagcatgaa agctactgcc aaggccaggt ggcccttctt 96540 ggttatagaa ggcaggcagt aaccagtaag taaccacatt tgtcaactgc caggagccag 96600 gcagaagttt cttggaggtc acccactccc aggagacaga gccttctctg ccctgtgctc 96660 caggcctcct gcagagcctt gtcttcggac cccagggtag gatattgcaa agcccacctg 96720 tcccctcagt acctctctga gtaagacccc tagaggacag gggagagcct cttgtgatct 96780 ctacaggcag gttagctgct tggccagctt ttacccaggg atgccctaac tgccttgcat 96840 cttagagggt agcagtacct gagccctcca tttgcggctc tcctgggtaa tggggtctgg 96900 caggtctggg tttgactcct ttcttaggaa atctcggaac tgactgaatc cctctcctta 96960 cgctaactcc aggagaagtc gaattcgtat cctactcaat tactaatgac aacaattttt 97020 gagtgcttac tatgtgagga ttcttattta attgatctaa taactcagta agttttaaaa 97080 agtattagcc tcatttttct gattaaaaaa gaaaagtggc caggtgcagt ggctcacacc 97140 tgtaatccca tcactttggg aggccaaggt aggaggattg cttgagtcca ggagtttaag 97200 accagcctga gcaacatagt gagacctcat ctctacaaaa aaaaaaaaaa aaaaaaaaaa 97260 aatttagcca ggcttggtgg cgtgcacctg tagtcccagc tactcagaag gctgaggcgg 97320 gaggatcact ttagcccagg aggtcaaggc tgcagtgagc catgattgtg ccactgtact 97380 ccagcctgga tgagagtgag accctgtctc aaaacaaaaa aagaaaaagg ctgggcatgg 97440 tggcacatgc ctataatcct agcactttgg gaggccaagg cgagtggatc acctgaggtc 97500 agcagttcaa gaccagcctg gccaagatgg tgaaactctg tctctactaa aaaaaaaata 97560 caaaaattag ccaggcatgg tggtgggcgc ctataatccc agctactcgg gaggctgaga 97620 cagagaattg cttgaacccg ggaggcagag gttgcagtga gccgagatcg tgccactgca 97680 ctccagcctg ggagacagag cgagactctg tctcaaaaaa aaaaaaagaa aagaaaaaga 97740 aaagtgaagg agtctgcccc aagtcacaaa gctagtaaac tgctgagctg gaactcaagc 97800 ccaggccttt gtcaccaaag tctgtcttac cccagctagc gtagctcagt cactcagtaa 97860 gttagagtcc ttgtgtgctg ggcactgtga acaaaacaga caaaatcact gctcttgcaa 97920 agatacacca aaaaaaaaaa aaaatcacat cttgtggagc ttgcgatctg gaggggccac 97980 cccatcattc ctaccttgct gactctagag cttctggggg ccttaggctc caaaaggatt 98040 tggcccatgc acctgtaaag ggatggggat gtcagaggtg ctggggcctg cctgggctcc 98100 ttgctgactg cccccttccc tgtgcagcta tccgttttgg tcggatgccg gaggctgaga 98160 agaggaagct ggtggcaggg ctgactgcaa acgaggggag ccagtacaac ccacaggtgg 98220 ccgacctgaa ggccttctcc aagcacatct acaatgccta cctgaaaaac ttcaacatga 98280 ccaaaaagaa ggcccgcagc atcctcaccg gcaaagccag ccacacggcg gtgagtgttg 98340 ctgctgcttg gcctggcagc atcctgggct ctgggtccca ctgccgcctg cctgactccg 98400 ggagagccag gccttctccc tccctcaact tcatggtgca ggcaagggac atggggagca 98460 cagggtgggg gtctcccgag gcctgatctc taacggggcc tggttttcag ccctttgtga 98520 tccacgacat cgagacattg tggcaggcag agaaggggct ggtgtggaag cagttggtga 98580 atggcctgcc tccctacaag gagatcagcg tgcacgtctt ctaccgctgc cagtgcacca 98640 cagtggagac cgtgcgggag ctcactgagt tcgccaagag catccccagc ttcagcagcc 98700 tcttcctcaa cgaccaggtt acccttctca agtatggcgt gcacgaggcc atcttcgcca 98760 tgctggcctc tatcgtcaac aaggacgggc tgctggtagc caacggcagt ggctttgtca 98820 cccgtgagtt cctgcgcagc ctccgcaaac ccttcagtga tatcattgag cctaagtttg 98880 aatttgctgt caagttcaac gccctggaac ttgatgacag tgacctggcc ctattcattg 98940 cggccatcat tctgtgtgga ggtgagtgag agtggggcag gtgggctggc ctggcacacc 99000 cagtcgtcct gggggttggc cctcactgca gggcactgtg cctgagctct gacagtgtgg 99060 ggaagtgtcc ctgtgatctt ggcagtggaa catgcaaggc actgactgag catgcaggat 99120 cagctccatc tcattatgta cgtagataga ggtggagaca ggaaaaagac taagccagac 99180 gtggtggctc acacctgtaa tcccagcact ttggcaggcc gaggcgggtg gatcacttga 99240 ggtcaggagt tcgaaaccag cctggccaac atggtgaaac cccgtctcta ctaaaaatac 99300 aaaaaattag ccagatgtgg tggcacgcgc ctgtaatccc agctacttgg gaggctgagc 99360 caggagaatc gcttgaaccc gagaggtgga ggttgcagtg agccaaaatc ccaccactgc 99420 actccagcct gggtgacaga gtgagaccct gtctcaaaaa aaaggaaaag gactaacagg 99480 cagtatgctg tcatgttaat gtggggtgga aaaattgtct gcattttttc tgcattttta 99540 aaattccaac acaataaata caataataac tatgctaact aacagtggtc tagagcttac 99600 ttcatgccag gcactgttct tttcatcgat gatgactcac ttgatcctca caacaaccct 99660 gtgcaggaag aatgttttgt gtctccattt tacacatcag agaggctgaa tgacctgcct 99720 atagcctcac aggcagacac aggatttgaa ttaagcattg agtctcttaa ccacaatact 99780 acgttgccta atcggggggg aggtggggac aaattggcaa aaaacaaaag aagtggatta 99840 agaccagggg tagggagatt agaacaccca gtggagcatt gctgatggga cagggcttgg 99900 tctgtcacgg ccaaggaggc ctgccgtccc ctgggccaag tcacctcttg gggtggaagt 99960 aggggagctc cactgccttt ctgagctccc tggcgtgccc tgtgtcccca cagaccggcc 100020 aggcctcatg aacgttccac gggtggaggc tatccaggac accatcctgc gtgccctcga 100080 attccacctg caggccaacc accctgatgc ccagtacctc ttccccaagc tgctgcagaa 100140 gatggctgac ctgcggcaac tggtcaccga gcacgcccag atgatgcagc ggatcaagaa 100200 gaccgaaacc gagacctcgc tgcaccctct gctccaggag atctacaagg acatgtacta 100260 acggcggcac ccaggcctcc ctgcagactc caatggggcc agcactggag gggcccaccc 100320 acatgacttt tccattgacc agcccttgag cacccggcct ggagcagcag agtcccacga 100380 tcgccctcag acacatgaca cccacggcct ctggctccct gtgccctctc tcccgcttcc 100440 tccagccagc tctcttcctg tctttgttgt ctccctcttt ctcagttcct ctttcttttc 100500 taattcctgt tgctctgttt cttcctttct gtaggtttct ctcttccctt ctcccttgcc 100560 ctccctttct ctctccaccc cccacgtctg tcctcctttc ttattctgtg agatgttttg 100620 tattatttca ccagcagcat agaacaggac ctctgctttt gcacaccttt tccccaggag 100680 cagaagagag tggggcctgc cctctgcccc atcattgcac ctgcaggctt aggtcctcac 100740 ttctgtctcc tgtcttcaga gcaaaagact tgagccatcc aaagaaacac taagctctct 100800 gggcctgggt tccagggaag gctaagcatg gcctggactg actgcagccc cctatagtca 100860 tggggtccct gctgcaaagg acagtgggca ggaggcccca ggctgagagc cagatgcctc 100920 cccaagactg tcattgcccc tccgatgctg aggccaccca ctgacccaac tgatcctgct 100980 ccagcagcac acctcagccc cactgacacc cagtgtcctt ccatcttcac actggtttgc 101040 caggccaatg ttgctgatgg ccccctgcac tggccgctgg acggcactct cccagcttgg 101100 aagtaggcag ggttccctcc aggtgggccc ccacctcact gaagaggagc aagtctcaag 101160 agaaggaggg gggattggtg gttggaggaa gcagcacacc caattctgcc cctaggactc 101220 ggggtctgag tcctggggtc aggccaggga gagctcgggg caggccttcc gccagcactc 101280 ccactgcccc cctgcccagt agcagccgcc cacattgtgt cagcatccag ggccagggcc 101340 tggcctcaca tccccctgct cctttctcta gctggctcca cgggagttca ggccccactc 101400 cccctgaagc tgcccctcca gcacacacac ataagcactg aaatcacttt acctgcaggc 101460 tccatgcacc tcccttccct ccctgaggca ggtgagaacc cagagagagg ggcctgcagg 101520 tgagcaggca gggctgggcc aggtctccgg ggaggcaggg gtcctgcagg tcctggtggg 101580 tcagcccagc acctgctccc agtgggagct tcccgggata aactgagcct gttcattctg 101640 atgtccattt gtcccaatag ctctactgcc ctccccttcc cctttactca gcccagctgg 101700 ccacctagaa gtctccctgc acagcctcta gtgtccgggg accttgtggg accagtccca 101760 caccgctggt ccctgccctc ccctgctccc aggttgaggt gcgctcacct cagagcaggg 101820 ccaaagcaca gctgggcatg ccatgtctga gcggcgcaga gccctccagg cctgcagggg 101880 caaggggctg gctggagtct cagagcacag aggtaggaga actggggttc aagcccaggc 101940 ttcctgggtc ctgcctggtc ctccctccca aggagccatt ctgtgtgtga ctctgggtgg 102000 aagtgcccag cccctgcccc tacgggcgct gcagcctccc ttccatgccc caggatcact 102060 ctctgctggc aggattcttc ccgctcccca cctacccagc tgatgggggt tggggtgctt 102120 cctttcaggc caaggctatg aagggacagc tgctgggacc cacctccccc tccccggcca 102180 catgccgcgt ccctgccccg acccgggtct ggtgctgagg atacagctct tctcagtgtc 102240 tgaacaatct ccaaaattga aatgtatatt tttgctagga gccccagctt cctgtgtttt 102300 taatataaat agtgtacaca gactgacgaa actttaaata aatgggaatt aaatatttaa 102360 gagctgactg gaagctgact cagttacttg catgtttttc ctggggctta cagggctcca 102420 cgcctcctcc acatccagta ctggagggca aaggaggctt tgggctccaa aaccctcccc 102480 tgcctccacc tcgctttgct caccgcttgt cagtcaggtg gacgactatg ccatttccgc 102540 cctgcagaga gaatttgggg tgtgagggga caaaggactt gtggtgccct ggcctcacct 102600 ggtggagcac ttggggtctg gggaagggga aggcccctgg aggaggcgga tgcaggactc 102660 aatagatcaa agccagtttt tcatcaccac aagagatcac ggctttcctc tcctttgctg 102720 ccacccagct ctctcctgtc tttcctgagt gccatctccc cagcggtcca gtcgagccca 102780 gcccccggca gccatgggtt tgtttgcagt gtgaggccag gtcagggtgt gtaacagagt 102840 atgtgtttag tcaggaaaaa actacagcta aatatttcca aatggggaat gtactgcagg 102900 gagttgttac aaaagtattg gaagtactga aagatcaata gggggaaata ggagtcatcc 102960 agagatgact aactgcagga agccattctc acctccaggg ctgaatggga aaatggtatt 103020 cagggcccac cgttgctaca acacacactg acactgcggc gacccaggag cctgaagtca 103080 ccagttgatt tgactgtaga accagactgc tggtgcctga gttcacgcag atggtacata 103140 tggggccacc agagtgcccg aggccaccac cactgctgcc cctggaacct cactgcttga 103200 gtgctgtagc cccataactc atagcctggt ctcagctgct tctgccagaa gtgctgtcag 103260 agtttggggt tggggaagca aaggattctt ccttctgcct atcttccagt ctgttgcttt 103320 tacctcccat tgggggaaaa aaatgcatgg tagtctagga aatggagttt gtagacttcc 103380 agcccttgca gcatagagga gcatgtggaa gggtgggaac tgaaccaagc gtctgcaggc 103440 aaacaactag cacagcgtac aggcaccagc cccagtgtga gggatcccag aggcccacag 103500 ctaagggatg gtcactacag cacttgcctc cttgttttct ttctttgctg gtcatcttgg 103560 cagagcaatc tgaattgtca ggtcccatat ggttcatctc agtccagaga gtgacagccc 103620 caggggaaca ggaggaaaag aggagaagga agaaaaaaag gggtgcttgg aaccagggca 103680 tctctgaaat tggtagcctt ttttataatg gaatcctctg gtttacttgg tgttataagt 103740 aaaatgttta ttcagaaaca gaatgcttgt tccttggaac tataaggaaa aattagcatt 103800 tagacaaaaa gttttctcag caaggcaatt ttactttctg caggaagggt gttcctcaca 103860 gatggaacaa tggcgagagc acacacgaac aaaggaggga agcaattttt atcctttatg 103920 cagcttgtcc ctgctactgt gtcctgtctc cattggctga agccagacca cacaatctaa 103980 gctaaacctg actggctaat aacttaaaac tttcctaaat aggtgaaggc aagggagaac 104040 aaaggaaaag aggaagttgc ttgcaaaagg acttagaaaa gtaataacca aatatctggt 104100 taaagtacaa ggacatagaa tgtactaatt cccttatatc taacagctac acaggatagg 104160 gcttaacaaa gagttattag cacaaaacaa ggtggcttga aggaagttag tctttaaaag 104220 aaactattat ttctaacact tatga 104245 5 22 DNA Artificial Sequence PCR Primer 5 accctgatgc ccagtacctc tt 22 6 23 DNA Artificial Sequence PCR Primer 6 gtctcggttt cggtcttctt gat 23 7 22 DNA Artificial Sequence PCR Probe 7 acctgcggca actggtcacc ga 22 8 19 DNA Artificial Sequence PCR Primer 8 gaaggtgaag gtcggagtc 19 9 20 DNA Artificial Sequence PCR Primer 9 gaagatggtg atgggatttc 20 10 20 DNA Artificial Sequence PCR Probe 10 caagcttccc gttctcagcc 20 11 10343 DNA M. musculus unsure 2359 unknown 11 tatcggactc ggtaccccca gggggccttg gtgatcagct agatagattc aggtcctgaa 60 tgaagctggg cttcctgggc aaaggtgttt ctgtttgcat gggatgcctt tgagctgcct 120 gtgggacgtg cataaggagg ggaccacgct ggagctcagg ctctgaacag ggctcccctg 180 agctttgaga agctgaagct gcactcttgc tattctctgc tgggtgagcc ctccacagcc 240 ctcccccacc ccaccccacc cgtagttgtc actctgagaa caggatttat gggcagtttt 300 ttatttgatg aaccttggag tagcatcagg gttactactg ctgaaatgag acacggtgac 360 cagatgcaac ctggggagga aagggtttat tcagctaata tttccatatc ctgctcatca 420 gaggaagtca gggcaggaac ctggaggcag gagctgatgt agaggccatg gagggtgctc 480 agcctgcttt cttatagaac ccaggaccag cccagggatg gcaccatcta ccaggggctg 540 ggccctcccc catcaatcat taattaagaa aatgcccttc agctggatct tatggaggca 600 ttttctcaat tgaggtgccc tcctttcaga ctccagactg tgtcaagttg acataaaact 660 aggcagtgcc cagcccctcc tggaacaatc ccgggaggac aagctggctc tgcagtctgt 720 ggcagccagc acagtgggag cgccgagctg ttctctaccc tcctacctca ggcaggacag 780 accactcctc agaacagcgg ttctggacag ctgcgtcctg agccatgggc tgggtggggg 840 gtgggctgcc ttgagtacca atcccccggg tgatcgctca caccagtgtt ccagcactga 900 gagcaaaggg acaggaggtt gaggtcatct ttggccgcat ggccaattca gtccatcctg 960 ggctttataa agctctgtct ccagaaacca gaaactgcag gcactgagag gggcacagga 1020 gcagcactgc tgtgtgacag tggccctgca gggtgcctgt gcccaccacc ttttccctga 1080 ccatctctgt ctctccctgc ccaggcagtc catctgcgct cagacccaga tggtggcaga 1140 gctatgacca ggcctgcagg cgccacgcca agtgggggtc agtcatggaa cagccacagg 1200 aggagacccc tgaggcccgg gaagaggaga aagaggaagt ggccatgggt gacggagccc 1260 cggagctcaa tgggggacca gaacacacgc ttccttccag cagctgtgca ggtatggagg 1320 gggctgagcc gggcggtggg ggttcaggtt tctgtgaaca acgtggggtg ccaagaagta 1380 gagtgactga tgctagagcc tggggccatg ctggtcacta gccagtgagc cgaggggccc 1440 tgggaaatca catacaggag tgtaggtgag ctgtacatga gcttttctgg gaggtgggaa 1500 ggtgttggtc ccttatccta cccctgccca tcagatgtct ctggccccac atgtgcacct 1560 gcacacgttt ggtaacaaac aggctttggt gccagagcct gtgcatgaga tagccttgtg 1620 tggacagaca ccacccctgg gatgcaccca tagtccaagc tgcactcggt gtcatggacc 1680 accggacccg ggagacctgg tactaaccct gtgctccgct atgtttgagc ggaaatgcca 1740 agtggtctga gtgagtgcct gtgccatggg ctgcttttga gcatcagggc tcagctggct 1800 cctcccactt gccgggtcac ttcatttagc ctccgttggt ggcaatggtg accttctctg 1860 gtagccagca tggccctgtc atctcatcct gcctgcacac agggcccctc aggcccctag 1920 atttccctcc tcagcctcgc ctcctctggc tgtcttgggc cagtgcatcc tggtgatcct 1980 ggatcggccc tcagcccagg gatgggcgtg gcttgctgct ggttgcagtt ggataggcca 2040 ctccaggggt acccaaggtc acagggctat ggggtaggat aaacctttgc ctgggcctgc 2100 aatcaagaag tggccaaatc cttgaaccat gtcacctgaa acctacagag caaggccttt 2160 cctctttagc actcagaggg tagggtggaa agaccagtgt ctgagagtcc agagtagccc 2220 atgtttttgt ctggctgtgg tgtgatggtc ctgcagagag agggtagctt ccaggatcca 2280 gaatgcactc agcactgtca caggctgctt ttacacaaca agggaccaga agcatggcag 2340 tgtgtgctgt taaccccanc actcangagg cagaggcang atgatcccgg agagttcaag 2400 gccagccctg gtctgtatag cctggacctt gccgagacag agaggggaga gggtnnnctn 2460 tnccnctntc ttttcctctc cttcctttcc cgtcgtcgcg cctccccgcc tccctctccg 2520 gctccccttc cccccccccc cgccctcgtt ccccccgngc ggcgttgcgc gctgcgggcc 2580 gtggccgcgg tcgcgcgttg tctggcggcc cggtcccgtc ccctcgcttg tcttgtgcgc 2640 tccctcggtc ccctccctgc ggccgcnggc cccctctcgc ccccccgtgc cctcctcccg 2700 ccccgcgccg cccgcccccg cggtccgccc gtcggctccg cggggggcgg gtcgcgccgc 2760 ggcgcgcgct cccctnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnctttt 2880 cgtgtttttg ggggtgaaga aaaacggggg acgaaggggg gcgagaggag agaaacgcac 2940 gggcacgcag gggggaacag gacgaacgag gcggaggggg gagagaanan cgggcggcgg 3000 ggacgccggg ggacaacgca gccgggaagg acccgacggg gggccggggc gcgcccgaac 3060 aacgcccgac cgcgaaaccg ggaccagggc gggcacggcc aacgagacgg aggggggggg 3120 ggggggggag ccgagcgagc ggagaaaggg agaggggacc gcggggagga ccgagcgggc 3180 gaagccgaga cgacaacaaa cggggaagga accggggcgg acgaacaggg cgggagggaa 3240 aaggggcggg ggcggaagaa cggggggggg cggggggggg gggggggggg aaacgcgcgg 3300 gcccggggag ggcgggcggg ggggccgggc cggggggagg gcgggggggg ggcggcgggg 3360 gcgggcgggg cggggggggg gcagagagag agagagagag agagagaacc cagacacatg 3420 tatataaaga aaaaaagaaa gaaagaaaga aagtaaaaac tttggaatta gaactaaatc 3480 cagagtgttc gtatgacctg tgaccccagt gctggggtta ggagacagga gacccctagg 3540 ctcactgctc agtcagccag gccaggcaga gagctcctgg tttggtggga aaccatgact 3600 caaaaaatac agtggaagtc atgcgtggtg tccatcacct ttaatcccag cactcaagag 3660 gcagaggcag gaggatctct gagtttgagg ccagtgtggt ctataaatct agttccagga 3720 caaccaggtc tatacagaga aactgtgtct caaaaagaaa ataaaagaaa aaaagacaac 3780 agtgaggagt gatacacaca cacacagaca cacacacaca cacacacaca catgcatgca 3840 tgttcaaggc tgacctcagt cacatagtaa attcaaaact agcctgggtc acaagagcta 3900 ttgtctcaaa aaaaaaaaaa aaaagaggaa agagagaatt caggaaggga aaatagtgcc 3960 cacagagcag acccaacctt ttgcaccctc gccctgaaga gcttggggca gtgttctgtg 4020 tgaagaaggg tcttatagaa tcccaacgtc cacttgtgca gtcaccgatc tgccacactc 4080 tgatcagggt gtaacagaga aggcacagga ccatcacctc caggagctgt gcctgcagtg 4140 gggtcattct cctgggtgtg atattgtggt ggtatgagga caaatcctca agccagtggc 4200 cagccaggaa gaagcagaaa cattgttttt ggaaaagcca cccagacact atttctatcc 4260 actcagcaca catactcaga tgacgccata cccgtcccca caggtcctgc ttctctggcc 4320 ttgtgtgtca agttacacat ggcttggagc cctcactcaa gctgaggcat tccaccactc 4380 actggtgagc tgggcctgtt tctctgtggt agtaattccc ctggcctacc tcgggtagcc 4440 agtaggacct ggttatcttg gctaatcaca gggttctgga ggtgtacaga caccccaccc 4500 catccccgag ccagagagca ctccaggaca gccatggatg tggggagaca agaggctgta 4560 ccgggtttct cctggccctg cctgcacagg aggggcttgc cgtggagagc acagacccag 4620 agagctgggg agcttgccat gagcttggta tccacaactt ctcagccttg ggcgtcagtg 4680 tgcccacatg agaacgcaca gaccccatcc ccatcccttg gtgaaactac caggaagtcc 4740 aaagctgctt taccctccag gagggcctgc cagagaaaat gaggggatca gctctcctca 4800 gctggggccg gagggcattt atacctttgg aatgacagtc gtcctgaact agggttccag 4860 gccacttatg accttatgta acacacgcaa actttagcgt ctgggatggc tatagccccc 4920 atggtccctt gctgcctgag ttcccctgct gtggccagtg tacaggctgg gagtgctggt 4980 ctgcgctgat cacgtggctg tggtctctct cctgcttgag tgtctctcac tggtctgggg 5040 agttggtgcc tgtctctgag gaagtgagtc tgcagcccca ggagccagcc cagactgtaa 5100 gtaaggccag caaacggaag tggatgggta gtactgtgaa gccagtctca tcgcttgtct 5160 cctccttaaa tctcaaagta gactcagaag aggctcgctg gcgtctagac agttgtgtga 5220 cagcaggtcc atccctgtga ccgtgcagtt acagcctccc actgtccttg ccagccttga 5280 aacctcagcc caaacctctg tgtacgcagt agaacagggc aagctggtgc tgcacctgag 5340 ctcactccag caggacttct gggagagtcc tgacagcaac cctggggtcc acagagactt 5400 gaactcctgg gatgggagcc agctgggcgg agctccgcac ccccacagtt cccaacagac 5460 acactccacc ccaccatcat ccgggaccca ctgaagatgc tatttaccca aagcccacca 5520 ggccccctcc tctcccagtc agggaggctg tgagcggcag ctgctcagct gcctgcctag 5580 cggggcttcc cccgaccctg aactggagag gaggctgggc ccttgaagct tttccccagt 5640 cacacagccc atggctgctg agctcttcag ctgagctcta ggctgcagct cttccttccc 5700 ccacctggct tgtaggcctg gtagctgatt gtttaactgc ccctccgtcc tgtcttcaca 5760 gacctctccc agaattcctc cccttcctcc ctgctggacc agctgcagat gggctgtgat 5820 ggggcctcag gcggcagcct caacatggaa tgtcgggtgt gcggggacaa ggcctcgggc 5880 ttccactacg gggtccacgc gtgcgagggg tgcaaggtac agatggactg aggggcgtgg 5940 ggatttgcct gcttcaggta ggaccccgaa aatagctcca gcaaagctct ttggttcctt 6000 agaggcctga gctctgggtc atccaaggga agaaaagaca ggtgtccaaa tggacacctg 6060 cctactgacc cagctatcca ctcagacgct gtggatggcc agctctgggg cctgggctcc 6120 agcaggcagg cagggaaaga gcaagcttgg gctgagtgtc cattgaagtc agtttccagg 6180 gccttgggtc ttaagaggat ctttggcaag agagtgatgc ttctaagttg ttctaacgac 6240 acaccatcag aatttcacaa agcgccatct gaaaggatgg cgagaggcgg ctcagtgctg 6300 aagagcgctt gctgctcttc tgaggatccg agtttgattt ccagcaacca tgtctggtgg 6360 ttcaatctcc tattacccca gctccagtcc tcttctggcc tgaagtcacc cacactcaac 6420 atgcgtgcac gtgcccacac acaggtacac atgcgcccac acgcaggcac acatgcatta 6480 aagtatatct ttcaaattgc catatggggt tggaaatgtg cttgcctgcc tgggagaagc 6540 ccttggtttg agcgacatca ccacaaaaac tgggtgtggt gctatacacg gtaatcttag 6600 ggatttggag ctaaaggcaa gaagatcagc tcaaagttgg gctggaaaga tggctcagcg 6660 gatagagctg ctgccttaag cctggtgacc cgagttccgt ttccaagaac caactggaaa 6720 gagggaacca attcttgtct gctggccttc acatgcatag ccccccacac cccccaaata 6780 aataaataaa taaataaatg ttaaataaac caagttctta tgaaaacaga agggttataa 6840 ttataaatta ttactaattt tgtttgtttt gttgagacgg ggttttactg tttagccctg 6900 gccatcctgg gactcactat gtaaactagg tggccttaaa ctcacaaaga tcctctgtct 6960 cccaagagct gggattaagg ccatggacca gtatacctag cccttaaatt tttttgggtg 7020 ctggaggaat ggcttagtgg ttaagaacac tgagtgcttt ccagaggttc tgagttcaat 7080 tcccagcaac cacatggtgg ctcacaacca tctgtgccgg gatccagtgc cctcttctgg 7140 tgtgtgtctg aagacaatga tggtgtgctc acataaataa aataaattaa ttttttaaat 7200 taaggatatt tttcttcttt cctatcccaa acaaacaaac aaaattcccc tggggtgtgg 7260 agagcaatct cattggttaa gagcactagc tactcttcca gaggacccag gatcaattcc 7320 cagtgcctac atggctgctc aaaacctttt gatacccctc acacatacat aaatgcaagc 7380 aaagcaacaa tgaaaataaa tcattttttt aaaaatattc cccatcactg ccaatttcag 7440 cctttttgtg accttacgtg ccactgggct ctggggcagc gcaggctaac gtccttggtc 7500 tcgcagggct tcttccgccg gacaatccgc atgaagctcg agtatgagaa gtgcgatcgg 7560 atctgcaaga tccagaagaa gaaccgcaac aagtgtcagt actgccgctt ccagaagtgc 7620 ctggcactcg gcatgtcgca caacggtgag ggcgcctgcg cagtctgtct caggacccca 7680 aagggcgcct gcgcagcaca gctaacccag ggtcccattc tgtcagaggg cgcccagcac 7740 ctgcccctgg cacggatggg tgtgtgcact tgctgttgat ggctgataca aatgccagga 7800 cctcagaggc ctgggccggc accagctagc tctctgtgcc tgtggctcac cctgttctgc 7860 ttcaagtgac aagcccagca gggatggggt tgccaacact gctccccaca cttgtgcctg 7920 acagcaggca aagctgaggc cagggttccc cggtcctgaa ctgnnnnnnn nnnnnnnnnn 7980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8040 nnnnnnnnnn nnnnnnnnnn nnnggtaccc agtcacctct gtttgcctct tcacccccag 8100 ctgtttcata tgagttcagt ggcctcaggt cctcaaggta ccactccagc ccgcacctct 8160 gcccagagtg aatgcccctg gttgtcctag taagtgcaaa ggatttccat tttccctgac 8220 ctacaaagac agggaaggaa gaaagccata tagccgggcg tggtggtgca tgcctttaat 8280 cccagcactc gagaggcaga ggcaggcaga tttctgagtt cgaggccagc ctggtctaca 8340 gagtgagttc caggatagcc agggctacac agatagaccc tgtctcagga aaaaaaaaaa 8400 aaaaaaaagc cataaaatgg aaaggtaaag agccggagct caggcatggg ccctggtcac 8460 cacaggatct ttctagttgc catgcctggc cagcccccgg cggagtcagc aaaggctctc 8520 atcatggagc tgcttccccg ctgactgtgg ggcctctggg ggcccctggc tcctaaagga 8580 tgatccccct cacctccaca cctgagagga gcaggggaga ggaggggtgc tggcccggcc 8640 ccccatgact ggctcccacc ccctatgcag ctatccgctt tggacggatg ccggaggccg 8700 agaagaggaa gctggtggcg gggctgactg ccagcgaggg gtgccagcac aacccccagc 8760 tggccgacct gaaggccttc tctaagcaca tctacaacgc ctacctgaaa aacttcaaca 8820 tgaccaaaaa gaaggcccgg agcatcctca ccggcaagtc cagccacaac gcagtgagtg 8880 tcactggcca gcccagccat gaggtggggt tcaggggaca cgagggtggg gtccccgtcc 8940 ttccccaggc ctggcctctg atgggcctgg ttttcagccc tttgtcatcc acgacatcga 9000 gacactgtgg caggcagaga agggcctggt gtggaaacag ctggtgaacg ggctgccgcc 9060 ctacaacgag atcagtgtgc acgtgttcta ccgctgccag tccaccacag tggagacagt 9120 ccgagagctc accgagttcg ccaagaacat ccccaacttc agcagcctct tcctcaatga 9180 ccaggtgacc ctcctcaagt atggcgtgca cgaggccatc tttgccatgc tggcctccat 9240 cgtcaacaaa gacgggctgc tggtggccaa cggcagtggc ttcgtcaccc acgagttctt 9300 gcgaagtctc cgcaagccct tcagtgacat cattgagccc aagttcgagt ttgctgtcaa 9360 gttcaatgcg ctggagctcg atgacagtga cctggcgctc ttcatcgcgg ccatcattct 9420 gtgtggaggt gagggggcgg agccgcactg ccagggacca aacctgacct cagacagtgt 9480 agcacagagc acatgtgcag aaccagctgc atctggttgg acagacagag gtgtaccttg 9540 ctgggattat agctggatgg cgggatgctt gtctagcaag cgggaggcgc tgagtccaat 9600 ccccagtact ggggcgtggg gaaaagaaat ccaccatttg tgggtagcgg agaaatttct 9660 gtgttacttt cacatttttc tagtccagta taatacacag ggaagcctgc tcccccacag 9720 gagcagagaa cacacctcag gccagaggtt tttaactcat ttggtcctca cagcaactgt 9780 gttaaggaca ctgaagccca gagccaccag gatgtccttc cacagagaca ggctcagaat 9840 ccgggtgcca ggtgtcctag ctaggaatca tgcagcccta tagatcagga aaaacaactg 9900 gtcagaaaca ggatgaatgg agtccccagg ggtggggtta gccacagagc aagacggtcc 9960 ctgcctgaga ttactcatac cccgtgtccc cacagaccgg ccaggcctca tgaatgtgcc 10020 ccaggtagaa gccatccagg acaccattct gcgggctcta gaattccatc tgcaggtcaa 10080 ccaccctgac agccagtacc tcttccccaa gctgctgcag aagatggcag acctgcggca 10140 gctggtcact gagcatgccc agatgatgca gtggctaaag aagacggaga gtgagacctt 10200 gctgcacccc ctgctccagg aaatctacaa ggacatgtac taaggccgca gcccaggcct 10260 cccctcaggc tctgctgggc ccagccacgg actgttcaga ggaccagcca caggcatggc 10320 aggggtaccg agtccgatat aag 10343 12 21 DNA Artificial Sequence PCR Primer 12 gtcatccacg acatcgagac a 21 13 19 DNA Artificial Sequence PCR Primer 13 gcccgttcac cagctgttt 19 14 21 DNA Artificial Sequence PCR Probe 14 tgtggcaggc agagaagggc c 21 15 20 DNA Artificial Sequence PCR Primer 15 ggcaaattca acggcacagt 20 16 20 DNA Artificial Sequence PCR Primer 16 gggtctcgct cctggaagat 20 17 27 DNA Artificial Sequence PCR Probe 17 aaggccgaga atgggaagct tgtcatc 27 18 3301 DNA H. sapiens unsure 2966 unknown 18 gaattctgcg gagcctgcgg gacggcggcg ggttggcccg taggcagccg ggacagtgtt 60 gtacagtgtt ttgggcatgc acgtgatact cacacagtgg cttctgctca ccaacagatg 120 aagacagatg caccaacgag ggtctggaat ggtctggagt ggtctggaaa gcagggtcag 180 atacccctgg aaaactgaag cccgtggagc aatgatctct acaggactgc ttcaaggctg 240 atgggaacca ccctgtagag gtccatctgc gttcagaccc agacgatgcc agagctatga 300 ctgggcctgc aggtgtggcg ccgaggggag atcagcc atg gag cag cca cag gag 355 Met Glu Gln Pro Gln Glu 1 5 gaa gcc cct gag gtc cgg gaa gag gag gag aaa gag gaa gtg gca gag 403 Glu Ala Pro Glu Val Arg Glu Glu Glu Glu Lys Glu Glu Val Ala Glu 10 15 20 gca gaa gga gcc cca gag ctc aat ggg gga cca cag cat gca ctt cct 451 Ala Glu Gly Ala Pro Glu Leu Asn Gly Gly Pro Gln His Ala Leu Pro 25 30 35 tcc agc agc tac aca gac ctc tcc cgg agc tcc tcg cca ccc tca ctg 499 Ser Ser Ser Tyr Thr Asp Leu Ser Arg Ser Ser Ser Pro Pro Ser Leu 40 45 50 ctg gac caa ctg cag atg ggc tgt gac ggg gcc tca tgc ggc agc ctc 547 Leu Asp Gln Leu Gln Met Gly Cys Asp Gly Ala Ser Cys Gly Ser Leu 55 60 65 70 aac atg gag tgc cgg gtg tgc ggg gac aag gca tcg ggc ttc cac tac 595 Asn Met Glu Cys Arg Val Cys Gly Asp Lys Ala Ser Gly Phe His Tyr 75 80 85 ggt gtt cat gca tgt gag ggg tgc aag ggc ttc ttc cgt cgt acg atc 643 Gly Val His Ala Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Thr Ile 90 95 100 cgc atg aag ctg gag tac gag aag tgt gag cgc agc tgc aag att cag 691 Arg Met Lys Leu Glu Tyr Glu Lys Cys Glu Arg Ser Cys Lys Ile Gln 105 110 115 aag aag aac cgc aac aag tgc cag tac tgc cgc ttc cag aag tgc ctg 739 Lys Lys Asn Arg Asn Lys Cys Gln Tyr Cys Arg Phe Gln Lys Cys Leu 120 125 130 gca ctg ggc atg tca cac aac gct atc cgt ttt ggt cgg atg ccg gag 787 Ala Leu Gly Met Ser His Asn Ala Ile Arg Phe Gly Arg Met Pro Glu 135 140 145 150 gct gag aag agg aag ctg gtg gca ggg ctg act gca aac gag ggg agc 835 Ala Glu Lys Arg Lys Leu Val Ala Gly Leu Thr Ala Asn Glu Gly Ser 155 160 165 cag tac aac cca cag gtg gcc gac ctg aag gcc ttc tcc aag cac atc 883 Gln Tyr Asn Pro Gln Val Ala Asp Leu Lys Ala Phe Ser Lys His Ile 170 175 180 tac aat gcc tac ctg aaa aac ttc aac atg acc aaa aag aag gcc cgc 931 Tyr Asn Ala Tyr Leu Lys Asn Phe Asn Met Thr Lys Lys Lys Ala Arg 185 190 195 agc atc ctc acc ggc aaa gcc agc cac acg gcg ccc ttt gtg atc cac 979 Ser Ile Leu Thr Gly Lys Ala Ser His Thr Ala Pro Phe Val Ile His 200 205 210 gac atc gag aca ttg tgg cag gca gag aag ggg ctg gtg tgg aag cag 1027 Asp Ile Glu Thr Leu Trp Gln Ala Glu Lys Gly Leu Val Trp Lys Gln 215 220 225 230 ttg gtg aat ggc ctg cct ccc tac aag gag atc agc gtg cac gtc ttc 1075 Leu Val Asn Gly Leu Pro Pro Tyr Lys Glu Ile Ser Val His Val Phe 235 240 245 tac cgc tgc cag tgc acc aca gtg gag acc gtg cgg gag ctc act gag 1123 Tyr Arg Cys Gln Cys Thr Thr Val Glu Thr Val Arg Glu Leu Thr Glu 250 255 260 ttc gcc aag agc atc ccc agc ttc agc agc ctc ttc ctc aac gac cag 1171 Phe Ala Lys Ser Ile Pro Ser Phe Ser Ser Leu Phe Leu Asn Asp Gln 265 270 275 gtt acc ctt ctc aag tat ggc gtg cac gag gcc atc ttc gcc atg ctg 1219 Val Thr Leu Leu Lys Tyr Gly Val His Glu Ala Ile Phe Ala Met Leu 280 285 290 gcc tct atc gtc aac aag gac ggg ctg ctg gta gcc aac ggc agt ggc 1267 Ala Ser Ile Val Asn Lys Asp Gly Leu Leu Val Ala Asn Gly Ser Gly 295 300 305 310 ttt gtc acc cgt gag ttc ctg cgc agc ctc cgc aaa ccc ttc agt gat 1315 Phe Val Thr Arg Glu Phe Leu Arg Ser Leu Arg Lys Pro Phe Ser Asp 315 320 325 atc att gag cct aag ttt gaa ttt gct gtc aag ttc aac gcc ctg gaa 1363 Ile Ile Glu Pro Lys Phe Glu Phe Ala Val Lys Phe Asn Ala Leu Glu 330 335 340 ctt gat gac agt gac ctg gcc cta ttc att gcg gcc atc att ctg tgt 1411 Leu Asp Asp Ser Asp Leu Ala Leu Phe Ile Ala Ala Ile Ile Leu Cys 345 350 355 gga gac cgg cca ggc ctc atg aac gtt cca cgg gtg gag gct atc cag 1459 Gly Asp Arg Pro Gly Leu Met Asn Val Pro Arg Val Glu Ala Ile Gln 360 365 370 gac acc atc ctg cgt gcc ctc gaa ttc cac ctg cag gcc aac cac cct 1507 Asp Thr Ile Leu Arg Ala Leu Glu Phe His Leu Gln Ala Asn His Pro 375 380 385 390 gat gcc cag tac ctc ttc ccc aag ctg ctg cag aag atg gct gac ctg 1555 Asp Ala Gln Tyr Leu Phe Pro Lys Leu Leu Gln Lys Met Ala Asp Leu 395 400 405 cgg caa ctg gtc acc gag cac gcc cag atg atg cag cgg atc aag aag 1603 Arg Gln Leu Val Thr Glu His Ala Gln Met Met Gln Arg Ile Lys Lys 410 415 420 acc gaa acc gag acc tcg ctg cac cct ctg ctc cag gag atc tac aag 1651 Thr Glu Thr Glu Thr Ser Leu His Pro Leu Leu Gln Glu Ile Tyr Lys 425 430 435 gac atg tac taa cggcggcacc caggcctccc tgcagactcc aatggggcca 1703 Asp Met Tyr 440 gcactggagg ggcccaccca catgactttt ccattgacca gctctcttcc tgtctttgtt 1763 gtctccctct ttctcagttc ctctttcttt tctaattcct gttgctctgt ttcttccttt 1823 ctgtaggttt ctctcttccc ttctcccttc tcccttgccc tccctttctc tctcctatcc 1883 ccacgtctgt cctcctttct tattctgtga gatgttttgt attatttcac cagcagcata 1943 gaacaggacc tctgcttttg cacacctttt ccccaggagc agaagagagt gggcctgccc 2003 tctgccccat cattgcacct gcaggcttag gtcctcactt ctgtctcctg tcttcagagc 2063 aaaagacttg agccatccaa agaaacacta agctctctgg gcctgggttc cagggaaggc 2123 taagcatggc ctggactgac tgcagccccc tatagtcatg gggtccctgc tgcaaaggac 2183 agtggcagac cccggcagta gagccgagat gcctccccaa gactgtcatt gcccctccga 2243 tcgtgaggcc acccactgac ccaatgatcc tctccagcag cacacctcag ccccactgac 2303 acccagtgtc cttccatctt cacactggtt tgccaggcca atgttgctga tggcccctcc 2363 agcacacaca cataagcact gaaatcactt tacctgcagg caccatgcac ctcccttccc 2423 tccctgaggc aggtgagaac ccagagagag gggcctgcag gtgagcaggc agggctgggc 2483 caggtctccg gggaggcagg ggtcctgcag gtcctggtgg gtcagcccag cacctcgccc 2543 agtgggagct tcccgggata aactgagcct gttcattctg atgtccattt gtcccaatag 2603 ctctactgcc ctccccttcc cctttactca gcccagctgg ccacctagaa gtctccctgc 2663 acagcctcta gtgtccgggg accttgtggg accagtccca caccgctggt ccctgccctc 2723 ccctgctccc aggttgaggt gcgctcacct cagagcaggg ccaaagcaca gctgggcatg 2783 ccatgtctga gcggcgcaga gccctccagg cctgcagggg caaggggctg gctggagtct 2843 cagagcacag aggtaggaga actggggttc aagcccaggc ttcctgggtc ctgcctggtc 2903 ctccctccca aggagccatt ctatgtgact ctgggtggaa gtgcccagcc cctgcctgac 2963 ggnnnnnnng atcactctct gctggcagga ttcttcccgc tccccaccta cccagctgat 3023 gggggttggg gtgcttcttt cagccaaggc tatgaaggga cagctgctgg gacccacctc 3083 cccccttccc cggccacatg ccgcgtccct gcccccaccc gggtctggtg ctgaggatac 3143 agctcttctc agtgtctgaa caatctccaa aattgaaatg tatatttttg ctaggagccc 3203 cagcttcctg tgtttttaat ataaatagtg tacacagact gacgaaactt taaataaatg 3263 ggaattaaat atttaaaaaa aaaagcggcc gcgaattc 3301 19 20 DNA Artificial Sequence Antisense Oligonucleotide 19 ccagggcagc agttgtaaga 20 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 tctgggtgct ccagtattgg 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21 tactccctcc cttttgcagt 20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 caagtagctg ggattacagg 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23 caatatgctt ctattaccag 20 24 20 DNA Artificial Sequence Antisense Oligonucleotide 24 tcctacaaca tctcagcctg 20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 tgctaattgt ttacacaata 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 agccctctgt gctcctggtc 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 tcagtttcac catctttgat 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28 gcagcaggca cgcgatagct 20 29 20 DNA Artificial Sequence Antisense Oligonucleotide 29 ctgtacaaca ctgtcccggc 20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 tcacgtgcat gcccaaaaca 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 tggtgagcag aagccactgt 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 ctgttggtga gcagaagcca 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33 catctgttgg tgagcagaag 20 34 20 DNA Artificial Sequence Antisense Oligonucleotide 34 ctcgttggtg catctgtctt 20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 cattccagac cctcgttggt 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 ttccagacca ctccagacca 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 ccatcagcct tgaagcagtc 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38 ttcccatcag ccttgaagca 20 39 20 DNA Artificial Sequence Antisense Oligonucleotide 39 gtctgaacgc agatggacct 20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 tggctgctcc atggctgatc 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 cgggagaggt ctgtgtagct 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 gtcacagccc atctgcagtt 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43 cactccatgt tgaggctgcc 20 44 20 DNA Artificial Sequence Antisense Oligonucleotide 44 acccggcact ccatgttgag 20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 ccacctgtgg gttgtactgg 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 agaaggcctt caggtcggcc 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 gcttggagaa ggccttcagg 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48 ttgtagatgt gcttggagaa 20 49 20 DNA Artificial Sequence Antisense Oligonucleotide 49 taggcattgt agatgtgctt 20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 tcaggtaggc attgtagatg 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 agtttttcag gtaggcattg 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 cgggccttct ttttggtcat 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53 tgaggaagag gctgctgaag 20 54 20 DNA Artificial Sequence Antisense Oligonucleotide 54 tggtcgttga ggaagaggct 20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 tcgtgcacgc catacttgag 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 atggcctcgt gcacgccata 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 gttggctacc agcagcccgt 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58 taggctcaat gatatcactg 20 59 20 DNA Artificial Sequence Antisense Oligonucleotide 59 ccacacagaa tgatggccgc 20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 cggtctccac acagaatgat 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 cctggccggt ctccacacag 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 atgaggcctg gccggtctcc 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63 acgttcatga ggcctggccg 20 64 20 DNA Artificial Sequence Antisense Oligonucleotide 64 ccatcttctg cagcagcttg 20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 ccgctgcatc atctgggcgt 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 gtgccgccgt tagtacatgt 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 caggaagaga gctggtcaat 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68 acaggaagag agctggtcaa 20 69 20 DNA Artificial Sequence Antisense Oligonucleotide 69 tcctgttcta tgctgctggt 20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 aggtgtgcaa aagcagaggt 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 ctcaagtctt ttgctctgaa 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 agtgtttctt tggatggctc 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73 gcccagagag cttagtgttt 20 74 20 DNA Artificial Sequence Antisense Oligonucleotide 74 actgtccttt gcagcaggga 20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 aaaccagtgt gaagatggaa 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 tcagcaacat tggcctggca 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 ccatcagcaa cattggcctg 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78 ggccatcagc aacattggcc 20 79 20 DNA Artificial Sequence Antisense Oligonucleotide 79 tgcatggtgc ctgcaggtaa 20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 gcaggcccct ctctctgggt 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 aggacctgca ggacccctgc 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 gaagctccca ctgggcgagg 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83 tcccgggaag ctcccactgg 20 84 20 DNA Artificial Sequence Antisense Oligonucleotide 84 tcagaatgaa caggctcagt 20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 gggacaaatg gacatcagaa 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 agagctattg ggacaaatgg 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87 ggagggcagt agagctattg 20 88 20 DNA Artificial Sequence Antisense Oligonucleotide 88 ggacactaga ggctgtgcag 20 89 20 DNA Artificial Sequence Antisense Oligonucleotide 89 gccctgctct gaggtgagcg 20 90 20 DNA Artificial Sequence Antisense Oligonucleotide 90 ctgcgccgct cagacatggc 20 91 20 DNA Artificial Sequence Antisense Oligonucleotide 91 aggaagcctg ggcttgaacc 20 92 20 DNA Artificial Sequence Antisense Oligonucleotide 92 acttccaccc agagtcacat 20 93 20 DNA Artificial Sequence Antisense Oligonucleotide 93 gagcgggaag aatcctgcca 20 94 20 DNA Artificial Sequence Antisense Oligonucleotide 94 tcatagcctt ggctgaaaga 20 95 20 DNA Artificial Sequence Antisense Oligonucleotide 95 gctcctagca aaaatataca 20 96 20 DNA Artificial Sequence Antisense Oligonucleotide 96 tcgtcagtct gtgtacacta 20 97 1542 DNA M. musculus CDS (58)...(1380) 97 agatggtggc agagctatga ccaggcctgc aggcgccacg ccaagtgggg gtcagtc 57 atg gaa cag cca cag gag gag acc cct gag gcc cgg gaa gag gag aaa 105 Met Glu Gln Pro Gln Glu Glu Thr Pro Glu Ala Arg Glu Glu Glu L ys 1 5 10 15 gag gaa gtg gcc atg ggt gac gga gcc ccg gag ctc aat ggg gga cca 153 Glu Glu Val Ala Met Gly Asp Gly Ala Pro Glu Leu Asn Gly Gly Pro 20 25 30 gaa cac acg ctt cct tcc agc agc tgt gca gac ctc tcc cag aat tcc 201 Glu His Thr Leu Pro Ser Ser Ser Cys Ala Asp Leu Ser Gln Asn Ser 35 40 45 tcc cct tcc tcc ctg ctg gac cag ctg cag atg ggc tgt gat ggg gcc 249 Ser Pro Ser Ser Leu Leu Asp Gln Leu Gln Met Gly Cys Asp Gly Ala 50 55 60 tca ggc ggc agc ctc aac atg gaa tgt cgg gtg tgc ggg gac aag gcc 297 Ser Gly Gly Ser Leu Asn Met Glu Cys Arg Val Cys Gly Asp Lys Ala 65 70 75 80 tcg ggc ttc cac tac ggg gtc cac gcg tgc gag ggg tgc aag ggc ttc 345 Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly Phe 85 90 95 ttc cgc cgg aca atc cgc atg aag ctc gag tat gag aag tgc gat cgg 393 Phe Arg Arg Thr Ile Arg Met Lys Leu Glu Tyr Glu Lys Cys Asp Arg 100 105 110 atc tgc aag atc cag aag aag aac cgc aac aag tgt cag tac tgc cgc 441 Ile Cys Lys Ile Gln Lys Lys Asn Arg Asn Lys Cys Gln Tyr Cys Arg 115 120 125 ttc cag aag tgc ctg gca ctc ggc atg tcg cac aac gct atc cgc ttt 489 Phe Gln Lys Cys Leu Ala Leu Gly Met Ser His Asn Ala Ile Arg Phe 130 135 140 gga cgg atg ccg gag gcc gag aag agg aag ctg gtg gcg ggg ctg act 537 Gly Arg Met Pro Glu Ala Glu Lys Arg Lys Leu Val Ala Gly Leu Thr 145 150 155 160 gcc agc gag ggg tgc cag cac aac ccc cag ctg gcc gac ctg aag gcc 585 Ala Ser Glu Gly Cys Gln His Asn Pro Gln Leu Ala Asp Leu Lys Ala 165 170 175 ttc tct aag cac atc tac aac gcc tac ctg aaa aac ttc aac atg acc 633 Phe Ser Lys His Ile Tyr Asn Ala Tyr Leu Lys Asn Phe Asn Met Thr 180 185 190 aaa aag aag gcc cgg agc atc ctc acc ggc aag tcc agc cac aac gca 681 Lys Lys Lys Ala Arg Ser Ile Leu Thr Gly Lys Ser Ser His Asn Ala 195 200 205 ccc ttt gtc atc cac gac atc gag aca ctg tgg cag gca gag aag ggc 729 Pro Phe Val Ile His Asp Ile Glu Thr Leu Trp Gln Ala Glu Lys Gly 210 215 220 ctg gtg tgg aaa cag ctg gtg aac ggg ctg ccg ccc tac aac gag atc 777 Leu Val Trp Lys Gln Leu Val Asn Gly Leu Pro Pro Tyr Asn Glu Ile 225 230 235 240 agt gtg cac gtg ttc tac cgc tgc cag tcc acc aca gtg gag aca gtc 825 Ser Val His Val Phe Tyr Arg Cys Gln Ser Thr Thr Val Glu Thr Val 245 250 255 cga gag ctc acc gag ttc gcc aag aac atc ccc aac ttc agc agc ctc 873 Arg Glu Leu Thr Glu Phe Ala Lys Asn Ile Pro Asn Phe Ser Ser Leu 260 265 270 ttc ctc aat gac cag gtg acc ctc ctc aag tat ggc gtg cac gag gcc 921 Phe Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu Ala 275 280 285 atc ttt gcc atg ctg gcc tcc atc gtc aac aaa gac ggg ctg ctg gtg 969 Ile Phe Ala Met Leu Ala Ser Ile Val Asn Lys Asp Gly Leu Leu Val 290 295 300 gcc aac ggc agt ggc ttc gtc acc cac gag ttc ttg cga agt ctc cgc 1017 Ala Asn Gly Ser Gly Phe Val Thr His Glu Phe Leu Arg Ser Leu Arg 305 310 315 320 aag ccc ttc agt gac atc att gag ccc aag ttc gag ttt gct gtc aag 1065 Lys Pro Phe Ser Asp Ile Ile Glu Pro Lys Phe Glu Phe Ala Val Lys 325 330 335 ttc aat gcg ctg gag ctc gat gac agt gac ctg gcg ctc ttc atc gcg 1113 Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Leu Phe Ile Ala 340 345 350 gcc atc att ctg tgt gga gac cgg cca ggc ctc atg aat gtg ccc cag 1161 Ala Ile Ile Leu Cys Gly Asp Arg Pro Gly Leu Met Asn Val Pro Gln 355 360 365 gta gaa gcc atc cag gac acc att ctg cgg gct cta gaa ttc cat ctg 1209 Val Glu Ala Ile Gln Asp Thr Ile Leu Arg Ala Leu Glu Phe His Leu 370 375 380 cag gtc aac cac cct gac agc cag tac ctc ttc ccc aag ctg ctg cag 1257 Gln Val Asn His Pro Asp Ser Gln Tyr Leu Phe Pro Lys Leu Leu Gln 385 390 395 400 aag atg gca gac ctg cgg cag ctg gtc act gag cat gcc cag atg atg 1305 Lys Met Ala Asp Leu Arg Gln Leu Val Thr Glu His Ala Gln Met Met 405 410 415 cag tgg cta aag aag acg gag agt gag acc ttg ctg cac ccc ctg ctc 1353 Gln Trp Leu Lys Lys Thr Glu Ser Glu Thr Leu Leu His Pro Leu Leu 420 425 430 cag gaa atc tac aag gac atg tac taa ggccgcagcc caggcctccc 1400 Gln Glu Ile Tyr Lys Asp Met Tyr 435 440 ctcaggctct gctgggccca gccacggact gttcagagga ccagccacag gcactggcag 1460 tcaagcagct agagcctact cacaacactc cagacacgtg gcccagactc tcccccaaca 1520 cccccacccc caccaacccc cc 1542 98 485 DNA M. musculus 98 aaagttttgg caggagctgg gggattctgc ggagcctgcg ggacggcggc agcggcgcga 60 gaggcggccg ggacagtgct gtgcagcggt gtgggtatgc gcatgggact cactcagagg 120 ctcctgctca ctgacagatg aagacaaacc cacggtaaag gcagtccatc tgcgctcaga 180 cccagatggt ggcagagcta tgaccaggcc tgcaggcgcc acgccaagtg ggggtcagtc 240 atggaacagc cacaggagga gacccctgag gcccgggaag aggagaaaga ggaagtggcc 300 atgggtgacg gagccccgga gctcaatggg ggaccaaaac aaacaaacaa acaagcaaac 360 aaaaaaacta cagtcaaaat ctaatttgaa aaatatttct gcctttatta ttacttattt 420 gattttgggc cctgggagaa tggactgagg tacataattt acattgcaaa gcagacccag 480 ggacg 485 99 1323 DNA M. musculus CDS (1)...(1323) 99 atg gaa cag cca cag gag gag acc cct gag gcc cgg gaa gag gag aaa 48 Met Glu Gln Pro Gln Glu Glu Thr Pro Glu Ala Arg Glu Glu Glu Lys 1 5 10 15 gag gaa gtg gcc atg ggt gac gga gcc ccg gag ctc aat ggg gga cca 96 Glu Glu Val Ala Met Gly Asp Gly Ala Pro Glu Leu Asn Gly Gly Pro 20 25 30 gaa cac acg ctt cct tcc agc agc tgt gca gac ctc tcc cag aat tcc 144 Glu His Thr Leu Pro Ser Ser Ser Cys Ala Asp Leu Ser Gln Asn Ser 35 40 45 tcc cct tcc tcc ctg ctg gac cag ctg cag atg ggc tgt gat ggg gcc 192 Ser Pro Ser Ser Leu Leu Asp Gln Leu Gln Met Gly Cys Asp Gly Ala 50 55 60 tca ggc ggc agc ctc aac atg gaa tgt cgg gtg tgc ggg gac aag gcc 240 Ser Gly Gly Ser Leu Asn Met Glu Cys Arg Val Cys Gly Asp Lys Ala 65 70 75 80 tcg ggc ttc cac tac ggg gtc cac gcg tgc gag ggg tgc aag ggc ttc 288 Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly Phe 85 90 95 ttc cgc cgg aca atc cgc atg aag ctc gag tat gag aag tgc gat cgg 336 Phe Arg Arg Thr Ile Arg Met Lys Leu Glu Tyr Glu Lys Cys Asp Arg 100 105 110 atc tgc aag atc cag aag aag aac cgc aac aag tgt cag tac tgc cgc 384 Ile Cys Lys Ile Gln Lys Lys Asn Arg Asn Lys Cys Gln Tyr Cys Arg 115 120 125 ttc cag aag tgc ctg gca ctc ggc atg tcg cac aac gct atc cgc ttt 432 Phe Gln Lys Cys Leu Ala Leu Gly Met Ser His Asn Ala Ile Arg Phe 130 135 140 gga cgg atg ccg gac ggc gag aag agg aag ctg gtg gcg ggg ctg act 480 Gly Arg Met Pro Asp Gly Glu Lys Arg Lys Leu Val Ala Gly Leu Thr 145 150 155 160 gcc agc gag ggg tgc cag cac aac ccc cag ctg gcc gac ctg aag gcc 528 Ala Ser Glu Gly Cys Gln His Asn Pro Gln Leu Ala Asp Leu Lys Ala 165 170 175 ttc tct aag cac atc tac aac gcc tac ctg aaa aac ttc aac atg acc 576 Phe Ser Lys His Ile Tyr Asn Ala Tyr Leu Lys Asn Phe Asn Met Thr 180 185 190 aaa aag aag gcc cgg agc atc ctc acc ggc aag tcc agc cac aac gca 624 Lys Lys Lys Ala Arg Ser Ile Leu Thr Gly Lys Ser Ser His Asn Ala 195 200 205 ccc ttt gtc atc cac gac atc gag aca ctg tgg cag gca gag aag ggc 672 Pro Phe Val Ile His Asp Ile Glu Thr Leu Trp Gln Ala Glu Lys Gly 210 215 220 ctg gtg tgg aaa cag ctg gtg aac ggg ctg ccg ccc tac aac gag atc 720 Leu Val Trp Lys Gln Leu Val Asn Gly Leu Pro Pro Tyr Asn Glu Ile 225 230 235 240 agt gtg cac gtg ttc tac cgc tgc cag tcc acc aca gtg gag aca gtc 768 Ser Val His Val Phe Tyr Arg Cys Gln Ser Thr Thr Val Glu Thr Val 245 250 255 cga gag ctc acc gag ttc gcc aag aac atc ccc aac ttc agc agc ctc 816 Arg Glu Leu Thr Glu Phe Ala Lys Asn Ile Pro Asn Phe Ser Ser Leu 260 265 270 ttc ctc aat gac cag gtg acc ctc ctc aag tat ggc gtg cac gag gcc 864 Phe Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu Ala 275 280 285 atc ttt gcc atg ctg gcc tcc atc gtc aac aaa gac ggg ctg ctg gtg 912 Ile Phe Ala Met Leu Ala Ser Ile Val Asn Lys Asp Gly Leu Leu Val 290 295 300 gcc aac ggc agt ggc ttc gtc acc cac gag ttc ttg cga agt ctc cgc 960 Ala Asn Gly Ser Gly Phe Val Thr His Glu Phe Leu Arg Ser Leu Arg 305 310 315 320 aag ccc ttc agt gac atc att gag ccc aag ttc gag ttt gct gtc aag 1008 Lys Pro Phe Ser Asp Ile Ile Glu Pro Lys Phe Glu Phe Ala Val Lys 325 330 335 ttc aat gcg ctg gag ctc gat gac agt gac ctg gcg ctc ttc atc gcg 1056 Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Leu Phe Ile Ala 340 345 350 gcc atc att ctg tgt gga gac cgg cca ggc ctc atg aat gtg ccc cag 1104 Ala Ile Ile Leu Cys Gly Asp Arg Pro Gly Leu Met Asn Val Pro Gln 355 360 365 gta gaa gcc atc cag gac acc att ctg cgg gct cta gaa ttc cat ctg 1152 Val Glu Ala Ile Gln Asp Thr Ile Leu Arg Ala Leu Glu Phe His Leu 370 375 380 cag gtc aac cac cct gac agc cag tac ctc ttc ccc aag ctg ctg cag 1200 Gln Val Asn His Pro Asp Ser Gln Tyr Leu Phe Pro Lys Leu Leu Gln 385 390 395 400 aag atg gca gac ctg cgg cag ctg gtc act gag cat gcc cag atg atg 1248 cag tgg cta aag aag acg gag agt gag acc ttg ctg cac ccc ctg ctc 1296 cag gaa atc tac aag gac atg tac taa 1323 Gln Glu Ile Tyr Lys Asp Met Tyr 405 100 20 DNA Artificial Sequence Antisense Oligonucleotide 100 tggtcatagc tctgccacca 20 101 20 DNA Artificial Sequence Antisense Oligonucleotide 101 ctgaccccca cttggcgtgg 20 102 20 DNA Artificial Sequence Antisense Oligonucleotide 102 cctgtggctg ttccatgact 20 103 20 DNA Artificial Sequence Antisense Oligonucleotide 103 tgggcccagc agagcctgag 20 104 20 DNA Artificial Sequence Antisense Oligonucleotide 104 tctgaacagt ccgtggctgg 20 105 20 DNA Artificial Sequence Antisense Oligonucleotide 105 gccagtgcct gtggctggtc 20 106 20 DNA Artificial Sequence Antisense Oligonucleotide 106 gttgtgagta ggctctagct 20 107 20 DNA Artificial Sequence Antisense Oligonucleotide 107 ccacgtgtct ggagtgttgt 20 108 20 DNA Artificial Sequence Antisense Oligonucleotide 108 tgcacagcac tgtcccggcc 20 109 20 DNA Artificial Sequence Antisense Oligonucleotide 109 tgtcttcatc tgtcagtgag 20 110 20 DNA Artificial Sequence Antisense Oligonucleotide 110 agcgcagatg gactgccttt 20 111 20 DNA Artificial Sequence Antisense Oligonucleotide 111 accatctggg tctgagcgca 20 112 20 DNA Artificial Sequence Antisense Oligonucleotide 112 tctcccaggg cccaaaatca 20 113 20 DNA Artificial Sequence Antisense Oligonucleotide 113 gtccctgggt ctgctttgca 20 114 20 DNA Artificial Sequence Antisense Oligonucleotide 114 tcctcctgtg gctgttccat 20 115 20 DNA Artificial Sequence Antisense Oligonucleotide 115 tcctcttccc gggcctcagg 20 116 20 DNA Artificial Sequence Antisense Oligonucleotide 116 ggccacttcc tctttctcct 20 117 20 DNA Artificial Sequence Antisense Oligonucleotide 117 gttctggtcc cccattgagc 20 118 20 DNA Artificial Sequence Antisense Oligonucleotide 118 gggagaggtc tgcacagctg 20 119 20 DNA Artificial Sequence Antisense Oligonucleotide 119 ggaattctgg gagaggtctg 20 120 20 DNA Artificial Sequence Antisense Oligonucleotide 120 ctggtccagc agggaggaag 20 121 20 DNA Artificial Sequence Antisense Oligonucleotide 121 tgcagctggt ccagcaggga 20 122 20 DNA Artificial Sequence Antisense Oligonucleotide 122 ccatctgcag ctggtccagc 20 123 20 DNA Artificial Sequence Antisense Oligonucleotide 123 acagcccatc tgcagctggt 20 124 20 DNA Artificial Sequence Antisense Oligonucleotide 124 ccatcacagc ccatctgcag 20 125 20 DNA Artificial Sequence Antisense Oligonucleotide 125 gaggccccat cacagcccat 20 126 20 DNA Artificial Sequence Antisense Oligonucleotide 126 cgacattcca tgttgaggct 20 127 20 DNA Artificial Sequence Antisense Oligonucleotide 127 cttcatgcgg attgtccggc 20 128 20 DNA Artificial Sequence Antisense Oligonucleotide 128 ttctcatact cgagcttcat 20 129 20 DNA Artificial Sequence Antisense Oligonucleotide 129 tgctggcacc cctcgctggc 20 130 20 DNA Artificial Sequence Antisense Oligonucleotide 130 cttcaggtcg gccagctggg 20 131 20 DNA Artificial Sequence Antisense Oligonucleotide 131 gaaggccttc aggtcggcca 20 132 20 DNA Artificial Sequence Antisense Oligonucleotide 132 gggccttctt tttggtcatg 20 133 20 DNA Artificial Sequence Antisense Oligonucleotide 133 atgctccggg ccttcttttt 20 134 20 DNA Artificial Sequence Antisense Oligonucleotide 134 tgaggatgct ccgggccttc 20 135 20 DNA Artificial Sequence Antisense Oligonucleotide 135 gacaaagggt gcgttgtggc 20 136 20 DNA Artificial Sequence Antisense Oligonucleotide 136 aggcccttct ctgcctgcca 20 137 20 DNA Artificial Sequence Antisense Oligonucleotide 137 tgatctcgtt gtagggcggc 20 138 20 DNA Artificial Sequence Antisense Oligonucleotide 138 gactggcagc ggtagaacac 20 139 20 DNA Artificial Sequence Antisense Oligonucleotide 139 tcttggcgaa ctcggtgagc 20 140 20 DNA Artificial Sequence Antisense Oligonucleotide 140 gatgttcttg gcgaactcgg 20 141 20 DNA Artificial Sequence Antisense Oligonucleotide 141 aagaggctgc tgaagttggg 20 142 20 DNA Artificial Sequence Antisense Oligonucleotide 142 cctcgtgcac gccatacttg 20 143 20 DNA Artificial Sequence Antisense Oligonucleotide 143 gatggcctcg tgcacgccat 20 144 20 DNA Artificial Sequence Antisense Oligonucleotide 144 agcatggcaa agatggcctc 20 145 20 DNA Artificial Sequence Antisense Oligonucleotide 145 aggccagcat ggcaaagatg 20 146 20 DNA Artificial Sequence Antisense Oligonucleotide 146 ctgccgttgg ccaccagcag 20 147 20 DNA Artificial Sequence Antisense Oligonucleotide 147 agccactgcc gttggccacc 20 148 20 DNA Artificial Sequence Antisense Oligonucleotide 148 gacgaagcca ctgccgttgg 20 149 20 DNA Artificial Sequence Antisense Oligonucleotide 149 tgggtgacga agccactgcc 20 150 20 DNA Artificial Sequence Antisense Oligonucleotide 150 cgcaagaact cgtgggtgac 20 151 20 DNA Artificial Sequence Antisense Oligonucleotide 151 gcttgcggag acttcgcaag 20 152 20 DNA Artificial Sequence Antisense Oligonucleotide 152 gtcactgaag ggcttgcgga 20 153 20 DNA Artificial Sequence Antisense Oligonucleotide 153 ctcaatgatg tcactgaagg 20 154 20 DNA Artificial Sequence Antisense Oligonucleotide 154 ctcgaacttg ggctcaatga 20 155 20 DNA Artificial Sequence Antisense Oligonucleotide 155 agaatgatgg ccgcgatgaa 20 156 20 DNA Artificial Sequence Antisense Oligonucleotide 156 ccggtctcca cacagaatga 20 157 20 DNA Artificial Sequence Antisense Oligonucleotide 157 atggtgtcct ggatggcttc 20 158 20 DNA Artificial Sequence Antisense Oligonucleotide 158 gcagatggaa ttctagagcc 20 159 20 DNA Artificial Sequence Antisense Oligonucleotide 159 gacctgcaga tggaattcta 20 160 20 DNA Artificial Sequence Antisense Oligonucleotide 160 tggttgacct gcagatggaa 20 161 20 DNA Artificial Sequence Antisense Oligonucleotide 161 gctgtcaggg tggttgacct 20 162 20 DNA Artificial Sequence Antisense Oligonucleotide 162 gggaagaggt actggctgtc 20 163 20 DNA Artificial Sequence Antisense Oligonucleotide 163 catctgggca tgctcagtga 20 164 20 DNA Artificial Sequence Antisense Oligonucleotide 164 gtctcactct ccgtcttctt 20 165 20 DNA Artificial Sequence Antisense Oligonucleotide 165 gcaaggtctc actctccgtc 20 166 20 DNA Artificial Sequence Antisense Oligonucleotide 166 gtgcagcaag gtctcactct 20 167 20 DNA Artificial Sequence Antisense Oligonucleotide 167 ccaggatgca ctggcccaag 20 168 20 DNA Artificial Sequence Antisense Oligonucleotide 168 tgggagaggt ctgtgaagac 20 169 20 DNA Artificial Sequence Antisense Oligonucleotide 169 cagtccatct gtaccttgca 20 170 20 DNA Artificial Sequence Antisense Oligonucleotide 170 aaagatcctc ttaagaccca 20 171 20 DNA Artificial Sequence Antisense Oligonucleotide 171 tgaccagggc ccatgcctga 20 172 20 DNA Artificial Sequence Antisense Oligonucleotide 172 aagcggatag ctgcataggg 20 173 20 DNA Artificial Sequence Antisense Oligonucleotide 173 tgacactcac tgcgttgtgg 20 174 20 DNA Artificial Sequence Antisense Oligonucleotide 174 tgacaaaggg ctgaaaacca 20 175 20 DNA H. sapiens 175 cctgtaatcc cagctacttg 20 176 20 DNA H. sapiens 176 ctggtaatag aagcatattg 20 177 20 DNA H. sapiens 177 caggctgaga tgttgtagga 20 178 20 DNA H. sapiens 178 tattgtgtaa acaattagca 20 179 20 DNA H. sapiens 179 gaccaggagc acagagggct 20 180 20 DNA H. sapiens 180 atcaaagatg gtgaaactga 20 181 20 DNA H. sapiens 181 agctatcgcg tgcctgctgc 20 182 20 DNA H. sapiens 182 gccgggacag tgttgtacag 20 183 20 DNA H. sapiens 183 tgttttgggc atgcacgtga 20 184 20 DNA H. sapiens 184 acagtggctt ctgctcacca 20 185 20 DNA H. sapiens 185 tggcttctgc tcaccaacag 20 186 20 DNA H. sapiens 186 cttctgctca ccaacagatg 20 187 20 DNA H. sapiens 187 aagacagatg caccaacgag 20 188 20 DNA H. sapiens 188 aggtccatct gcgttcagac 20 189 20 DNA H. sapiens 189 gatcagccat ggagcagcca 20 190 20 DNA H. sapiens 190 aactgcagat gggctgtgac 20 191 20 DNA H. sapiens 191 ggcagcctca acatggagtg 20 192 20 DNA H. sapiens 192 ctcaacatgg agtgccgggt 20 193 20 DNA H. sapiens 193 ccagtacaac ccacaggtgg 20 194 20 DNA H. sapiens 194 ggccgacctg aaggccttct 20 195 20 DNA H. sapiens 195 cctgaaggcc ttctccaagc 20 196 20 DNA H. sapiens 196 ttctccaagc acatctacaa 20 197 20 DNA H. sapiens 197 aagcacatct acaatgccta 20 198 20 DNA H. sapiens 198 catctacaat gcctacctga 20 199 20 DNA H. sapiens 199 caatgcctac ctgaaaaact 20 200 20 DNA H. sapiens 200 atgaccaaaa agaaggcccg 20 201 20 DNA H. sapiens 201 cttcagcagc ctcttcctca 20 202 20 DNA H. sapiens 202 agcctcttcc tcaacgacca 20 203 20 DNA H. sapiens 203 ctcaagtatg gcgtgcacga 20 204 20 DNA H. sapiens 204 tatggcgtgc acgaggccat 20 205 20 DNA H. sapiens 205 acgggctgct ggtagccaac 20 206 20 DNA H. sapiens 206 cagtgatatc attgagccta 20 207 20 DNA H. sapiens 207 gcggccatca ttctgtgtgg 20 208 20 DNA H. sapiens 208 atcattctgt gtggagaccg 20 209 20 DNA H. sapiens 209 ctgtgtggag accggccagg 20 210 20 DNA H. sapiens 210 ggagaccggc caggcctcat 20 211 20 DNA H. sapiens 211 cggccaggcc tcatgaacgt 20 212 20 DNA H. sapiens 212 caagctgctg cagaagatgg 20 213 20 DNA H. sapiens 213 acgcccagat gatgcagcgg 20 214 20 DNA H. sapiens 214 acatgtacta acggcggcac 20 215 20 DNA H. sapiens 215 accagcagca tagaacagga 20 216 20 DNA H. sapiens 216 acctctgctt ttgcacacct 20 217 20 DNA H. sapiens 217 ttcagagcaa aagacttgag 20 218 20 DNA H. sapiens 218 gagccatcca aagaaacact 20 219 20 DNA H. sapiens 219 aaacactaag ctctctgggc 20 220 20 DNA H. sapiens 220 tccctgctgc aaaggacagt 20 221 20 DNA H. sapiens 221 ttccatcttc acactggttt 20 222 20 DNA H. sapiens 222 tgccaggcca atgttgctga 20 223 20 DNA H. sapiens 223 caggccaatg ttgctgatgg 20 224 20 DNA H. sapiens 224 ggccaatgtt gctgatggcc 20 225 20 DNA H. sapiens 225 acccagagag aggggcctgc 20 226 20 DNA H. sapiens 226 gcaggggtcc tgcaggtcct 20 227 20 DNA H. sapiens 227 cctcgcccag tgggagcttc 20 228 20 DNA H. sapiens 228 ccagtgggag cttcccggga 20 229 20 DNA H. sapiens 229 actgagcctg ttcattctga 20 230 20 DNA H. sapiens 230 ttctgatgtc catttgtccc 20 231 20 DNA H. sapiens 231 ccatttgtcc caatagctct 20 232 20 DNA H. sapiens 232 caatagctct actgccctcc 20 233 20 DNA H. sapiens 233 ctgcacagcc tctagtgtcc 20 234 20 DNA H. sapiens 234 cgctcacctc agagcagggc 20 235 20 DNA H. sapiens 235 gccatgtctg agcggcgcag 20 236 20 DNA H. sapiens 236 ggttcaagcc caggcttcct 20 237 20 DNA H. sapiens 237 atgtgactct gggtggaagt 20 238 20 DNA H. sapiens 238 tggcaggatt cttcccgctc 20 239 20 DNA H. sapiens 239 tgtatatttt tgctaggagc 20 240 20 DNA H. sapiens 240 tagtgtacac agactgacga 20 241 20 DNA M. musculus 241 tggtggcaga gctatgacca 20 242 20 DNA M. musculus 242 ccacgccaag tgggggtcag 20 243 20 DNA M. musculus 243 agtcatggaa cagccacagg 20 244 20 DNA M. musculus 244 ctcaggctct gctgggccca 20 245 20 DNA M. musculus 245 ccagccacgg actgttcaga 20 246 20 DNA M. musculus 246 gaccagccac aggcactggc 20 247 20 DNA M. musculus 247 agctagagcc tactcacaac 20 248 20 DNA M. musculus 248 acaacactcc agacacgtgg 20 249 20 DNA M. musculus 249 ggccgggaca gtgctgtgca 20 250 20 DNA M. musculus 250 ctcactgaca gatgaagaca 20 251 20 DNA M. musculus 251 aaaggcagtc catctgcgct 20 252 20 DNA M. musculus 252 tgcgctcaga cccagatggt 20 253 20 DNA M. musculus 253 atggaacagc cacaggagga 20 254 20 DNA M. musculus 254 cctgaggccc gggaagagga 20 255 20 DNA M. musculus 255 aggagaaaga ggaagtggcc 20 256 20 DNA M. musculus 256 gctcaatggg ggaccagaac 20 257 20 DNA M. musculus 257 cagctgtgca gacctctccc 20 258 20 DNA M. musculus 258 cagacctctc ccagaattcc 20 259 20 DNA M. musculus 259 cttcctccct gctggaccag 20 260 20 DNA M. musculus 260 tccctgctgg accagctgca 20 261 20 DNA M. musculus 261 gctggaccag ctgcagatgg 20 262 20 DNA M. musculus 262 ctgcagatgg gctgtgatgg 20 263 20 DNA M. musculus 263 atgggctgtg atggggcctc 20 264 20 DNA M. musculus 264 agcctcaaca tggaatgtcg 20 265 20 DNA M. musculus 265 gccggacaat ccgcatgaag 20 266 20 DNA M. musculus 266 atgaagctcg agtatgagaa 20 267 20 DNA M. musculus 267 gccagcgagg ggtgccagca 20 268 20 DNA M. musculus 268 cccagctggc cgacctgaag 20 269 20 DNA M. musculus 269 gaaggcccgg agcatcctca 20 270 20 DNA M. musculus 270 gccacaacgc accctttgtc 20 271 20 DNA M. musculus 271 tggcaggcag agaagggcct 20 272 20 DNA M. musculus 272 gccgccctac aacgagatca 20 273 20 DNA M. musculus 273 gctcaccgag ttcgccaaga 20 274 20 DNA M. musculus 274 ccgagttcgc caagaacatc 20 275 20 DNA M. musculus 275 caagtatggc gtgcacgagg 20 276 20 DNA M. musculus 276 atggcgtgca cgaggccatc 20 277 20 DNA M. musculus 277 ctgctggtgg ccaacggcag 20 278 20 DNA M. musculus 278 ggtggccaac ggcagtggct 20 279 20 DNA M. musculus 279 ccaacggcag tggcttcgtc 20 280 20 DNA M. musculus 280 ggcagtggct tcgtcaccca 20 281 20 DNA M. musculus 281 gtcacccacg agttcttgcg 20 282 20 DNA M. musculus 282 tccgcaagcc cttcagtgac 20 283 20 DNA M. musculus 283 ccttcagtga catcattgag 20 284 20 DNA M. musculus 284 tcattgagcc caagttcgag 20 285 20 DNA M. musculus 285 tcattctgtg tggagaccgg 20 286 20 DNA M. musculus 286 gaagccatcc aggacaccat 20 287 20 DNA M. musculus 287 ggctctagaa ttccatctgc 20 288 20 DNA M. musculus 288 aggtcaacca ccctgacagc 20 289 20 DNA M. musculus 289 gacagccagt acctcttccc 20 290 20 DNA M. musculus 290 aagaagacgg agagtgagac 20 291 20 DNA M. musculus 291 gacggagagt gagaccttgc 20 292 20 DNA M. musculus 292 agagtgagac cttgctgcac 20 293 20 DNA M. musculus 293 cttgggccag tgcatcctgg 20 294 20 DNA M. musculus 294 tgcaaggtac agatggactg 20 295 20 DNA M. musculus 295 tgggtcttaa gaggatcttt 20 296 20 DNA M. musculus 296 tcaggcatgg gccctggtca 20 

What is claimed is:
 1. An antisense oligonucleotide 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding PPAR-delta, wherein said antisense oligonucleotide specifically hybridizes with said nucleic acid molecule encoding PPAR-delta and has a sequence comprising SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 95 or
 96. 2. The antisense oligonucleotide of claim 1 which comprises at least one modified internucleoside linkage.
 3. The antisense oligonucleotide of claim 2 wherein the modified internucleoside linkage is a phosphorothioate linkage.
 4. The antisense oligonucleotide of claim 1 which comprises at least one modified sugar moiety.
 5. The antisense oligonucleotide of claim 4 wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 6. The antisense oligonucleotide of claim 1 which comprises at least one modified nucleobase.
 7. The antisense oligonucleotide of claim 6 wherein the modified nucleobase is a 5-methylcytosine.
 8. The antisense oligonucleotide of claim 1 which is a chimeric oligonucleotide.
 9. A compound 8 to 80 nucleobases in length which specifically hybridizes with at least an 8-nucleobase portion of a preferred target region on a nucleic acid molecule encoding PPAR-delta.
 10. A composition comprising the antisense oligonucleotide of claim 1 and a pharmaceutically acceptable carrier or diluent.
 11. The composition of claim 10 further comprising a colloidal dispersion system. 